Bio Lab 2 midterm
what is the equation for %error
%error=( |measured-expected|/ expected) *100
has education beed effective in limiting the spread of HIV/AIDS?
yes
what are the major proteins unique to milk?
caseins and whey proteins
what is the purified antigen
chicken gamma globulin
How could the presence or amount of specific proteins be measured
chromatography, SDS-PAGE, western blotting
tips for the P-20 are what color and what microliter amount is used?
clear: .5-.20
a gene (bla) that
codes for a protein that gives the bacteria resistance to an antibiotic.
in the Bradford Assay the intensity of the blue correlates with what? measured by?
concentration of protein; qualitatively by eyer, quantitatively with a spectrophotometer
Secondary antibody specifically recognizes the
constant region of the primary antibody
TE (elution) Buffer
remove GFP from the column
hydrophobic interaction chromatography step 1
-Hydrophobic proteins interact with column -Salt ions interact with the less hydrophobic proteins and H2O---
hydrophobic interaction chromatography step 2
-Less hydrophobic E. coli proteins fall from column -GFP remains bound to the column
hydrophobic interaction chromatography step 3
-Released from column matrix -Flows through the column
1micro litter is how many ml?
.001 ml
how many are unaware of their infection?
1 in 7
what are limitations of the Bradford Assay
1. the assay measures total protein concentration, different methods must be used to identify specific proteins 2.Assay is linear over a limited range 3.the coomassie dye bind specifically to arginine and hydrophobic amino acids 4.the Amino Adid composition can alter the concentration- absorbance curve. Use of a standards (like BSA- Bovine Serum Albumin) with a similar composition must be used.
hydrophobic interaction chromatography steps
1.Add bacterial lysate to column matrix in high salt buffer 2. Wash less hydrophobic proteins from column in low salt buffer 3.Elute GFP from column with no salt buffer
what are some ways that diseases spread?
1.Exchange of bodily fluid, ingestion of contaminated food of water, inhalation vector transfer
the average amino acid is how many daltons?
110
p-20 (174) =
17.4(upside nl)
When was HIV first diagnosed?
1981
half of all new infections are in people younger than what?
25
In the bradford assay what does the absorbance shift from?
470nm (reddish brown) to 595 nm (blue)
p-200 (057)=
57 (upside down nl)
p-1000 (097)=
970 (upside down nl)
Transformation efficiency
= Total number of cells growing on the agar plate/ Amount of DNA spread on the agar plate 190/0.16= 1.2*10^3 transformants/ng
what is a plasmid
A circular piece of autonomously replicating DNA Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest usually contains genes for one or more traits that may be beneficial to bacterial survival
f the sample gave a negative result for the disease-causing agent, does this mean that you do not have the disease? What reasons could there be for a negative result when you actually do have the disease?
A negative result does not necessarily mean that you do not have the disease. It could be a false negative. The ELISA may not be sensitive enough to detect very low levels of disease agent, such as the levels that might be present soon after infection. Another cause of false negatives is experimental error, such as putting a negative control into a well where you thought you were putting an experimental sample.
Restriction enzyme definition
A protein that recognizes a particular sequence of DNA and cuts the DNA at that site (the restriction site)
whats is Beers law equation
A=Ebc;
How can fragments of DNA be separated from one another?
Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their sizes. DNA is an acid and has many negative electrical charges. Scientists have used this fact to design a method that can be used to separate pieces of DNA. A solution containing a mixture of DNA fragments of variable sizes is placed into a small well formed in an agarose gel that has a texture similar to gelatin. An electric current causes the negatively-charged DNA molecules to move towards the positive electrode.The larger the size of the particles, however, the slower they are strained through the gel. After a period of exposure to the electrical current, the DNA fragments will sort themselves out by size. Fragments that are the same size will tend to move together through the gel and form bands.
4. Nutrient broth incubation reason
Allows beta-lactamase expression
why did you assay your samples in triplicate?
Assaying the samples in triplicate is another control. If you do not get the same result in all triplicate wells, you have a problem with your experimental technique or you have made a pipetting error. In a clinical laboratory, the experiment would have to be repeated. If this error occurs in this activity, take the result of the two matching wells since this is probably correct.
Gene expression
Beta Lactamase Ampicillin resistance Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription
which buffers have highest to least salt content
Binding, equilibration, wash, elution
why use column chromatography?
Chromatography used for protein purification Size exclusion Ion exchange Hydrophobic interaction
the ELISA kit results in what
Clear Determination Of Positive And Negative Results
What are the GFP purification kit advantages?
Cloning in action Links to biomanufacturing Biopharmaceutical development Amazing visual results
From the results that you obtained, how could you prove that these changes that occurred were due to the procedure that you performed?
Compare the control plates. cells that weren't tratedwith the plasmid (negs) could not grow ampicillin. The cells that were treated with the plasmid (posts) can grow on the LB/amp plate. Thus the plasmid must confer resistance to ampicillin.
Why do you need to assay positive and negative control samples as well as your experimental samples?
Controls are needed to make sure that the experiment worked. If there are no positive controls and the sample is negative, we can't know if the sample was truly negative or if the assay didn't work. Conversely, without a negative control, there is no way of knowing if all samples (positive or not) would have given a positive result.
The Bradford Assay uses what kind of dye?
Coomassie Blue dye
Determine the total amount of DNA in experiment
DNA (µg) = (concentration of DNA (µg/µl) x (volume of DNA in µl) 0.08* 10= 0.8 ng (upside down n)
DNA-
DNA-RNA-PROTEIN-TRAIT
changes in DNA lead to proteins with what?
Different functions, Novel traits, positive/ negative/ no effects
How do doctors use the immune response to protect you from disease?
Doctors use the immune response when we are vaccinated against diseases. Our immune system remembers the pathogens to which we have been exposed, and the next time we are exposed to the pathogens our immune system attacks them more quickly and efficiently. Doctors take advantage of this priming effect by exposing us to inactivated pathogens (killed or weakened organisms that cannot make us sick) so that if we are later exposed to the live pathogen, our body will mount a strong and immediate antibody response, reducing or eliminating the chance that it will make us sick.
ingestion of contaminated food of water ex
E.coli, prions that cause Creutzfeldt-Jakob and mad cow diseases, protozoa that cause giardiasis, nematodes that cause trichinosis
what are the methods of transformation and their definitions?
Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock
what does ELISA stand for?
Enzyme- Linked- Immunosorbant- Assay
Why are enzymes used in this immunoassay?
Enzymes provide a way to see whether the primary antibody has attached to its target (antigen) in the microplate well. Primary and secondary antibodies are invisible, so a detection method is necessary. The enzyme HRP is linked to the secondary antibody. HRP reacts with a colorless substrate in a chemical reaction that turns blue. If the secondary antibody is present in the well, the color change indicates a positive result.
Why is rapid detection of disease exposure important?
For many diseases, detecting the infection and beginning treatment early may reduce the severity of the symptoms or even prevent the disease completely and to prevent further spread of the disease.
Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate
Fraction of DNA used = Volume spread on LB/amp plate/ Total volume in test tube: 100/510= .2
sample with elution buffer
GFP elutes from the columns, GFP travels down the column as a ring and elutes into tube
What does GFP stand for? What is pglo and where did it come from
Green Fluorescent Protein; the pGLO transformation kit, use a procedure to transform bacteria with a gene that codes for(GFP). bioluminescent jellyfish Aequorea victoria, and GFP causes the jellyfish to fluoresce and glow in the dark.
HIV is treated but not cured by drugs which inhibits the action of what?
HIV enzymes
what is the real world and objective of tracking outbreaks of disease?
HIV, Bird Flu and West Nile viruses, common cold, cholera, smallpox, anthrax, and STDs; Epidemiology, disease spread, public health
what is the real world and objective of detecting antibodies in serum?
HIV, Lyme disease, trichinosis, West Nile virus, and Bird Flu virus; Detecting exposure to disease causing agents
Exchange of bodily fluid ex
HIV, SARS, Epstein-Barr virus (cause of mononucleosis), STDs
If you tested positive for disease exposure, did you have direct contact with one of the original infected students? If not, what conclusions can you reach about transmissibility of disease in a population?
Having intimate contact with another person means that you are exposed to any germs that a person may have contracted from any previous intimate contacts.
What does HIV stand for?
Human Immunodeficiency Virus
Restriction enzyme digestion is used for...
Identifying individuals (DNA fingerprinting) Identifying species (e.g. Mytilus) Cloning (moving genes in and out of plasmids)
Can you predict what would happen if you took one of the green colonies from the LB/amp/ara plate and streaked it onto an LB/amp plate? Conversely, what would hap- pen if you took a white colony from the LB/amp plate and streaked it onto an LB/amp/ara plate? Explain your answer.
If a green colony was streaked onto a LB/amp plate the colonies would be white bc this plate does not contain arabinose which is needed to induce expression of the GFP gene and generate green fluorescent colonies. If a white colony was streaked onto a LB/amp/ara plate it would be green bc This plate contains arabinose which induces expression of the GFP gene and generates green fluorescent colonies.
When you added secondary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen?
If the sample contained antigen, the secondary antibody bound to the primary antibodies already bound to antigen in the wells. If the test sample did not contain antigen, primary antibody did not bind in the wells, so the secondary antibody had nothing to bind and was flushed out in the wash step.
When you added primary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen?
If the sample contained the antigen, the primary antibody bound the antigen. If it did not contain the antigen, the primary antibody did not bind and was flushed out in the wash step.
Why are immunosuppressant drugs necessary when someone has an organ transplant?
Immunosuppressive drugs (like prednisolone and cyclosporine) prevent the body from treating the transplanted organ as a foreign invader; Organs are rejected when the body mounts a strong immune response to the transplant. On the negative side, the action of immunosuppressive drugs is not specific and they suppress all immunological reactions. As a result, transplant recipients are very vulnerable to infections.
Genetic transformation is used in many areas of biotechnology
In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person's cells with healthy copies of the defective gene that causes the disease.
3. Heat-shock reason
Increases permeability of membranes
Transcriptional Regulation
Lactose operon Arabinose operon pGLO plasmid
What is nutrient broth?
Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression
vector transfer ex
Mosquito-borne diseases (malaria, West Nile virus, dengue fever, yellow fever), tick-borne diseases (Lyme disease, Rocky Mountain spotted fever)
Of the E. coli traits you originally noted, which seem now to be significantly different after performing the transformation procedure?
New trait (observed change) color ( LB/amp/ara colonies are green under UV) ampicillin ( transformed colonies can grow on ampicillin resistance)
Describe how you might recover the cancer-curing protein from the bacterial cells.
One can isolate a single fluorescent green colony of bacteria and grow large amounts of the bacteria in a liquid growth media. Bacteria in liquid media can be concentrated by centrifugation. After the bacterial cells are lysed to release the cancer curing protein, the protein can be isolated by passage through a chro- matography column which has an affinity for the cancer-curing protein.
Which of the traits that you originally observed for E. coli did not seem to become altered?
Original trait (analysis of observations) color (bacteria white) colony size (similar size)
what is the primary antibody
Polyclonal anti-chicken antibody made by rabbits
what is the secondary antibody?
Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP)
Transformation solution = CaCI2 reason
Positive charge of Ca++ ions shields negative charge of DNA phosphates
what is the real world and objective of detecting antigens?
Pregnancy, drug, GMO and allergen tests Air food and water testing HIV, smallpox, West niles and bird flu viruses; Uses for antibodies in research, medicine, and consumer goods
after 4-8 weeks of exposure to the HIV virus the body will have what?
Produced a detectable level of antibodies (immune response) against HIV
HIV is a ?
RNA retrovirus
restriction enzymes recognize what? and are isolated from what?
Recognize short specific sequences, often palindromes Isolated from bacteria as endogenous "restrictors" of bacterial pathogens
what is the transformation procedure?
Suspend bacterial colonies in Transformation solution Add pGLO plasmid DNA Place tubes on ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates
what does HIV infect?
T-cells in the immune system and thus destroys the immune system
What antibody-based tests can you buy at your local pharmacy?
Test kits that are based on the same principles as the ELISA include home pregnancy and ovulation tests, and tests for the presence of illegal drugs such as marijuana and cocaine.
restriction site
The actual sequence of DNA is called a
How does the immune system protect us from disease?
The immune system includes physical barriers, such as the skin and mucous membranes that prevent pathogens from entering the body, and cellular responses, such as circulating macrophages that respond to foreign invaders. Our acquired immune system mounts a specific antibody response when the body is exposed to a foreign invader, and our immune cells attack the invader.
ELISA (HIV test) detects what?
The presence of serum antibodies agains t HIV protein antigens
why use chromatography?
To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products.
what are the types of ELISA?
Tracking outbreaks of disease, detecting antigens, detecting antibodies in serum
what is in the Laemmli Buffer?
Tris buffe, SDS (sodium dodecyl Sulfate) detergent, Glycerol, Bromophenol Blue dye
What is transformation?
Uptake of foreign DNA, often a circular plasmid, change caused by genes and involves the insertion of one or more gene(s) into an organism in order to change the organism's traits.
inhalation ex
Viruses that cause the flu, bacteria that cause tuberculosis
2. Why did you need to wash the wells after every step?
Washing removes any proteins that have not bound to the plastic wells and any antibodies that have not bound to their targets, thus preventing unbound proteins (either antigen or antibodies) from giving false positive results.
what is a protein
a macromolecule which consists of side chains of Amino Acids
what is SDS detergent used for?
to dissolve proteins and give them a negative charge
To generate the standard curve, the measured
absorbance of each standard in the curve is plotted against the known protein concentration.
What is an example of a disease that attacks the human immune system?
autoimmune diseases (e.g., rheumatoid arthritis, lupus, asthma, eczema, SCID) and AIDS.
tips for the P-1000 are what color and what microliter
blue; 200-1000
What is a bacterial colony?
bacteria cells that came from a single clonal cell
why did you discard the supernatant of the protein purification procedure
bc it contains the bacterial growth media but not the desired GFP
what are the major whey proteins in cows milk and why are they important?
beta- lactoglobulin and alpha-lactalbumin which are important for lactose synthesis
how to do protein size comparison?
break protein complexes into individual proteins, denature proteins using detergent and heat, separate proteins based on size
how is HIV transmitted?
by exchange of body fluids, sharing needles, or blood transfusion
tips for the p-200 are what color and what microliter
yellow; .20-200
restriction enzymes how function
cut upend destroy invading DNA,Some restriction enzymes may leave a short length of unpaired nucleotide bases, called a "sticky" end, at the DNA site where they cut, whereas other restriction enzymes make a cut across both strands creating double stranded DNA fragments with "blunt" ends. cut the palindromes at restriction sites • Restriction enzymes recognize specific palindromes
cloning definition
duplication and propagation of a cell or organism
Can the assay be used qualitatively
yes
Lysozyme
enzymatically digest the bacterial cell wall, which weakens it so it erupts when frozen
in this lab students will use absorbance data from a set of protein samples with known concentrations to create a standard curve on linear graph paper. what can then be calculated?
estimate the proteinconcentration
-pGlo LB
even lawn of bacteria, white
genetic diversity provides pool for natural selection=
evolution
what are the symptoms of HIV>
flu like symptoms within 1-2 months followed by latent period of up to 10 years
why are caseins important?
for the growth and development of the nursing young
how may have HIV been spread?
form an animal host to humans
freezer
freeze the bacteria so the cytoplasm expands and ruptures the cell wall
By using a dilution series of known proteins, one can
generate a spectrophotometric standard curve.
explain the relationship between genes and proteins
genes contain the genetic code which determine the AA composition of a protein
3 examples of proteins found in your body
hormones, hemoglobins, antibodies
HIV as a virus replicates and
host cells are destroyed (lymphocytes) and the host immune system is crippled and enter the disease AIDS
When would a qualitative assay be acceptable vs. the quantitative assay?
if a spectrophotometer is not available, students can compare their unknown samples qualitatively to a dilution series to determine the protein concentrations.
how do you know if you were successful in isolation and purifying GFP from the cloned bacterial cells?
if tube 3 was green
what are ELISA test based on?
immune system antibody molecules
what are the other proteins found in milk?
immunoglobulins (antibodies), serum albumin, enzymes, growth factors, nutrient transporters
the samples that you added to the microplate strip contain many proteins and may or may not contain the disease antigen. What happened to the proteins in the plastic well if the sample contained the antigen? What if it did not contain the antigen?
in either case, all the proteins present in the sample bind to the plastic wells.
what is the purpose of rupturing/lysing the bacteria?
in order to release the GFP which can then be purified using column chromatography
antibodies are blank against HIV
ineffective
GFP links to real world
is a visual marker Study of biological processes (example: synthesis of proteins) Localization and regulation of gene expression Cell movement Cell fate during development Formation of different organs Screenable marker to identify transgenic organisms
what are the protein size measured in?
kD kilodaltons
shaking incubator importance
keeps bacteria warm and provides aeration which oxygenates bacterial cultures. Increased oxygen accelerated the growth of bacteria cause
incubator importance
keeps warm so bacteria will grow
how are bacterial cells in a library different from the cells in a colony
library has a diverse mixture of bacterial types which contain diverse mixture of gene. A colon originates from an individual clone which only contains a single gene
+pGlo, LB/amp
many colonies (75) white
+pGlo amp/ara
many colonies (75) white when exposed to room light but green when exposed to UV light
Colorimetric assays use standard curves created by
measuring the absorbances of solutions of known concentration to determine the concentration of unknown samples.
micropipettes are used to measure these minute
microliter (nl)(upside down n) volumes
very tiny amounts of chemical and biological reagents are used in many
molecular and cellular biology experiments
how many micrograms of DNA did you spread on the LB/amp/ara plates?
multiply the total amount of DNA used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. .8*.2= 0.16
is coating needed in microplane strips ?
no
-pGlo LB/amp
nothing grew
dalton is approximately the mass of
one hydrogen atom or 1.66*10^-24 gram
how many death due to HIV?
over 20 million deaths worldwide, over half a million in the US
how many currently infected with HIV?
over 40 million, more than 1.2 million in the US.
centrifuge
pellet the bacteria and separate it from the growth media
Wash buffer contains what? in order to?
phosphate buffer saline (PBS) to keep antibodies in a stable environment that helps keep their structure
Microplate strips are made of what?
polystyrene
Be familiar with common uses for ELISA today.
pregnancy tests, disease detection in people, animals, and plants, detecting illegal drug use, testing indoor air quality, and determining if food is labeled accurately
equilibrium buffer
prepares column for application of GFP. raises the salt concentration of the column to match the GFP
DNA encodes for what?
proteins that confer traits
what are reagents?
purified antigen, primary antibody (serum samples), secondary antibody (enzyme linked), enzyme substrates (3,3',5,5')
binding buffer
raises the salt concentration of GFP which causes a conformational change in GFP exposing the hydrophobic regions
Antibody will only bind to what?
simulated HIV antigen
Incubate on ice reason
slows fluid cell membrane
we measure absorbance by what?
spectrophotometers
sample in wash buffer
stick to columns, GFP remains as band at the top
sample in binding buffer
stick to columns, GFP remains as bands at the top
Proteins determine what ?
structure and function
traits are the result of?
structure and function
what is the enzyme substrate?
tetramethylbenzidine (TMB) - a colorless solution that when oxidized by HRP turns blue
the standard curve can then be used to estimate
the / concentration of protein in an unknown sample, based upon the measured absorbance value
pGLO plasmid encodes
the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells
explain why both the liquid cultures fluoresce green
the green colony is green bc the arabinose continues the expression of the GFP gene. The white colony is green bc the arabinose turns on the expression of the GFP gene.
UV light helpful
the green fluorescents and is visible under UV light
What color was the pellet in this step of the experiment? What color was the super- natant? What does this tell you?
the pallet was white or pale green bc it released the GFP from bacteria when it was lysed. The supernatant was fluorescent green bc the GFP remain in it
If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what can be inferred about the other genes on the plasmid that were involved in your transformation procedure?
the plasmid must express a gene for ampicillin resistance ( the protein product of the bra gene codes for beta- lactase, the protein breaks down the ampicillin)
Hydrophobic side chains in amino acids bind to what?
the polystyrene wells
Secondary antibody (enzyme-linked antibody) will only bind to
the primary antibody (serum antibody)
what do you analyze by performing the SDS-PAGE?
the specific protein content in the samples
why did you discard the white liquid from the - tube but keep the green one
the white culture of bacteria does not have the GFP and is not needed for purification step
What problems can prevent the immune system from working properly?
three categories: hypersensitivity, immunodeficiency, and autoimmune diseases. Hypersensitivity occurs when the immune system overreacts to an antigen; hypersensitivity reactions include anaphylactic reactions, allergies, and contact sensitivity (e.g., reaction to poison ivy). Immuno- deficiency means that an individual cannot mount an effective immune response. Immunodeficiency may be genetic (e.g., SCID or "bubble boy" disease) or induced by a disease (e.g., immunodeficiency from HIV infection) or by immunosuppressive drugs (e.g., drugs given after organ transplant to prevent rejection). Autoimmune disease results from the immune system inappropriately mounting an immune response to itself, for example, diseases like lupus, rheumatoid arthritis, multiple sclerosis (MS), insulin-dependent diabetes, and celiac disease.
Why heat the samples before putting them on the gels?
to denature the proteins complexes allowing the separation of individual proteins by size
Green Fluorescent Protein causes the jellyfish
to fluoresce and glow in the dark. 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.
what is glycerol used for?
to make samples sink into wells
What is the tris buffer used for?
to provide appropriate pH
If a solute absorbs light of a particular wavelength the absorbance is directly proportional to what?
to the concentration of that solute in solution up to a point
In Bradford Assay the coomassie blue dye binds to what?
to the side chains of specific amino acids
What is bromophenol blue dye used for?
to visualize samples
describe hydrophobic interaction chromatography and its purpose in lab.
used to separate or purify protein from other molecules. lab uses it to purify GFP based on its hydrophobic properties
wash buffer
wash away less hydrophobic contaminating proteins from the column
can you explain why the bacterial cells' outer cell wall ruptures when the cells are frozen? What happens to an unopened soft drink when it freezes?
when bacterial cell freezes the volume of the cytoplasm expands which puts pressure on the cell wall which ruptures
