Bio 230 LAB Exam 3
What is a "protein molecular weight standard" and what role does in serve in SDS-PAGE of various proteins?
4-20% gradient gel used and this gives a complete resolution of all bonds and resolves the broadly expressed proteins in E Coli
When GFP absorbs UV light does it re-emit light of a longer or shorter wavelength?
Absorbs it then re-emits light of a LONGER wavelength
What role does the polyacrylamide gel play in SDS-PAGE - how does it allow one to differentially separate proteins?
Acts as an ideal substrate through a solid sieving matrix
What effect does SDS have upon unequal charge distribution?
Acts to equalize unequal charge by coating and binding to proteins, penetrating interior and disrupting quaternary, tertiary, & secondary structures
Briefly describe the overall ELISA procedure as performed in lab.
Add samples - incubate, add primary - incubate, add secondary - incubate, add substrate - incubate
Briefly describe the significance of the 10-minute incubation or 'recovery' period post-heat shock transformation
Allows the cells to recover and express the ampicillin resistance protein beta lactamase (so transformed cells survive on ampicillin plates)
Briefly describe the structural relationship between antigen (epitope) and the antigen-binding sites?
Amino acids
What antibiotic is used in the selection process?
Ampicillin
What were the specific identities of the following ELISA 'reagents': purified antigen, primary antibody, and secondary antibody?
Antigen- chicken proteins Primary- polyclonal anti-chicken antibody made by rabbits Secondary- polyclonal anti-rabbit antibody made by goats lined to HRP
Differentiate between "antigens" and "epitopes" (antigenic determinants) - which is 'bigger'?
Antigens are portions of cells, viruses, and molecules that the body recognizes as foreign Epitopes are also known as 'antigenic determinants', because they are the actual part of an antigen that determines an immune response Antigen is larger
What is the "araC" gene product and what role does it play in bacterial transformation - be specific
AraC is a gene regulation system that controls the expression of the fluorescent protein in transformed cells
Which type of lymphocytes mediate the main response of humoral immunity?
B cells
What are the two types of lymphocytes involved in adaptive immunity?
B lymphocytes" (or 'B cells')arise and mature in the bone marrow of adults T lymphocytes (or 'T cells') begin in the bone marrow but mature in the thymus
What is the "bla" gene product and what role does it play in bacterial transformation?
B-lactamase - Confers resistance to antibiotic ampicillin
What affect does B-lactamase activity have upon ampicillin, and thus, upon transformed cells?
B-lactamase allows transformed cells the ability to grow on ampicillin plates
What is B-lactamase and what role does it play in bacterial transformation?
B-lactamase is a protein that inactivates the ampicillin present in ampicillin containing LB nutrient agar
Briefly describe the arabinose operon - how related genes for the catabolism of arabinose are expressed?
Bacterial genes that make enzymes to break down arabinose are not expressed when arabinose is absent from the environment. When arabinose is present, the genes are turned on
Why are ELISAs called "immunoassays"?
Because they are antibody-based, ELISAs
How is the transformation solution thought to facilitate the introduction of extracellular DNA into competent cells?
Ca2+ cation neutralizes the negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells
What is the chemical composition of the transformation solution?
Calcium Chloride
Differentiate between luminescence and fluorescence
Calcium-activated luminescent protein, which transfers energy to GFP, resulting in the fluorescence of the protein
What is the molecular nature of the GFP chromophore?
Cloning and expression of recombinant protein in E. Coli; it's fluorescent
What is SDS - how is it amphiphilic?
Current is applied to proteins in a solution and the charged properties allow them to be carried through an electric field
What effect do SDS, DTT, and a 950C heat treatment for five minutes have upon GFP activity?
Denatures GFP
What effect does SDS have upon quaternary, tertiary, and secondary protein structures(collectively)?
Disrupt them to change/equalize unequal charges
Name one bacterial species that is not naturally competent
E. coli
Briefly describe the overall concept of an enzyme-linked immunosorbent assay.
ELISA relies on antibodies to detect the presence of antigens in liquid samples
What does ELISA stand for?
Enzyme-Linked Immunosorbent Assay
What effect does cyclization have upon GFP chromophore activity?
Forms an active chromophore
Draw a simple diagram of the pGLO plasmid indicating the three principal genes that are present
GFP: - fluorescence B-lactamase: - confers resistance to antibiotic amphicillin Ara C Regulator protein: - controls expression of fluorescent protein in transformed cells
Briefly describe the 'continuous' polyacrylamide gel technique
In a 'CONTINUOUS SYSTEM', a gradient of acrylamide is used across the entire gel, typically starting with 4% and ending with 20%- With a gradient system, proteins of various sizes can be separated on a single gel type, providing greater flexibility and even separation over a broad molecular weight range
Describe Frederick Griffith's experiments with Streptococcus pneumoniae that led to the discovery of transformation
In virulent strains, each bacterium was surrounded with a polysaccharide capsule, which makes it appear smooth when grown on a plate. Griffith found that these virulent forms sometimes mutated to non-virulent forms, which appeared rough when grown on the plate. Under different conditions and using mice, Griffith found that the rough bacteria somehow acquired the genteel smooth type of bacteria that killed mice.
What is the proposed affect of "heat shock" upon membrane permeability?
It increases the permeability of the cell membrane
What is "horseradish peroxidase" (HRP), where is it 'found' in an ELISA, and what role does it serve in antigen detection?
It is attached to the secondary antibody and in the presence of TMB it produces a blue color
What role does the sugar arabinose play in the pGLO system?
It turns on the gene for GFP when present in the cell's nutrient medium
What is "aequorin" and what role does it play with respect to GFP fluorescence in vivo?
Its a calcium activated luminescent protein which transfers energy to GFP resulting in fluorescence
pGLO Bacterial Transformation
LAB 1
SDS-PAGE
Lab 2
Enzyme-linked Immunosorbent
Lab 3
What effect does the above (absorption and re-emission) have upon energetics?
Lower energy emitted and longer wavelength visible
Briefly describe the overall concept of adaptive immunity.
Lymphocytes which are a type of leukocyte('white blood cell') that acts against specific pathogens
Describe the significance or function of the following components of Laemmli sample buffer: Tris, glycerol, and bromophenol blue
Maintain protein at pH conductive to electrophoretic separation
What is the major function of B cells during the humoral immune response?
Make antibodies against antigens
What is a "cloning vector"?
Move genes from one organism to another
How is the GFP fluorophore activated in the absence of a luminescent protein?
Must have an external energy source to excite it
Differentiate between natural transformation and artificial transformation?
Natural: - Transformation occurs naturally, competence, cells have surface proteins that bind to DNA and transport it into the cell, homologous recombination Artificial: - Not naturally competent, chemical transformation to make bacteria competent, heat shock
Are all proteins equally charged or do they vary in their net charge?
Net negative charge, but individually vary
What is "homologous recombination - what role does it play in natural or artificial transformation?
New DNA can be substituted for the homologous region of the bacterium's chromosome
Are all bacteria naturally competent?
No
What were the expected/observed results and their significance?
No color change in negative control, color change in positive change due to presence of antigen.
With regard to chemical transformation, what is "heat shock" and how is it performed?
Once a bacterium is suspended in calcium chloride solution, the heat shock allows DNA molecules to be taken up by the cell
What is "chemical transformation" and how does it make the recipient bacteria competent?
Requires treating bacterial cells with high concentration of calcium chloride to make them competent
What structural feature of Streptococcus pneumoniae conferred virulence (or the lack thereof) in Griffith's transformation studies?
S type = smooth or virulence R type = rough or nonvirulent
What is "selection" and how are transformed cells selected via the pGLO system?
Selection for cells that have been transformed with pGLO DNA is accomplished by growth on anti-biotic plates
If net charge and protein structure are normalized via SDS, by what principle then will the proteins be separated during electrophoresis?
Size
What does "SDS-PAGE" stand for?
Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis
List two to three applications of ELISAs.
Testing for West Nile virus, HIV, SARS virus, hormones such as "human chorionic gonadotropin" (hCG), and drug screening.
With respect to transformation, what does it mean to be "competent"?
The ability of a bacterium to take up 'naked' DNA molecules
Differentiate between "primary" and "secondary" antibodies with respect to form (structure)and function
The primary antibodies recognize antigens, the secondary antibodies bind to primary antibodies
What are "antigen-binding sites"?
The variable regions form the antigen-binding site. These sites are complementary in shape to the three-dimensional shape of an epitope and bind precisely to it. This exact match creates the antibody specificity
Where are the "variable regions" found in your figure?
They are the arms of the chains and vary in amino acid sequence
What is the significance of the heavy and light variable regions in the context of forming a variety of antigen binding sites?
They can vary in amino acids thus able to bind to different epitopes
Briefly describe how naturally competent bacteria obtain 'naked' DNA from the environment- how did the DNA become part of the environment in the first place?
They have surface proteins that bind to DNA in the environment and transport it into the cell
Briefly describe the overall objective of the pGLO Bacterial Transformation system as used in lab
To transform bacteria with a 'plasmid' containing a gene that codes for Green Fluorescent Protein (GFP)
What affect does HRP have upon its substrate (tetramethylbenzidine)?
Turns blue
With respect to the above question, differentiate between "heavy" and "light" chains - how many of each make up a single antibody?
Two identical heavy chains, and two identical light chains. The heavy ones run the entire length of the antibody.
Describe the outcome of Griffith's final experiment in which both heat-killed S type and living R type bacteria were injected into a mouse and how this led him to his 'principle of transformation'
Type R bacteria had somehow been transformed into type S bacteria, acquiring the genetic virulence
How was GFP detected after SDS-PAGE of total protein extracts from transformants platedon LBamp + ara?
Under a UV light
What effect do SDS and DTT have upon GFP activity?
partial denatures GFP
What are "plasma cells", how do they arise, and what is their dedicated function?
When the epitopes attach to the receptors the stimulated B cell becomes a plasma cell which then secretes soluble antibody molecules.
What is the name of the arabinose operon regulatory protein
araC regulatory protein
What is an "ori" (origin of replication) and what role does it serve with respect to the plasmid?
- Ori is place where DNA replication begins - Allows plasmid to reproduce itself
What was the difference between Griffith's "S type" and "R type" bacteria, and what affect did this have on the mortality of his test mice?
- S type was virulent and surrounded with a polysaccharide cap, causing mice death - R type were nonvirulent forms that lacked a polysaccharide cap and did not cause disease
What are plasmids - how are they similar or different from the bacterial chromosome?
- Small circular pieces of DNA - Usually contain genes for one or more traits that may be beneficial to bacterial survival
Briefly describe the tertiary structure of GFP and the relative location of the fluorescent chromophore
- The tertiary structure of GFP is barrel-like, consisting of 11 beta sheets - Disulfide bonds between cysteine residues also contribute to a protein's tertiary structure.
What does "DTT" stand for, and what affect does it have upon disulfide bonds
- 1,4-Dithiothreitol - Reduces disulfide bonds by donating a H+ atom to S groups of cysteine
What is GFP - where did it 'come from'?
Green Fluorescent Protein Bioluminescent jellyfish: Aequorea victoria
Of the above, which is targeted towards extracellular pathogens?
Humoral
With respect to adaptive immunity, differentiate between humoral and cell-mediated responses
Humoral immune responses involve the descendants of B cells and the soluble proteins they secrete, called antibodies Cell-mediated immune responses involve T cells that regulate adaptive immune responses or attack intracellular pathogens
Of the above, which are used to directly recognize antigens?
Primary
What is the significance of the above upon protein structure?
Proteins would stay linear without it and won't migrate based on size
What is a "B cell receptor", where is it localized to in B cells, and what role does it play in humoral immunity?
Receptors that attach to the epitopes, they are localized as integral to the cytoplasmic membrane
Briefly describe the overall concept of SDS-PAGE
Used to separate the entire repertoire of proteins expressed in E. coli, which includes the foreign GFP protein
What are the temperature and temporal (time) parameters of the heat shock step?
Water bath is set at 42 degrees Celsius and for 50 seconds