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BIOL 3581 Lecture 9: Biotech Lecture 9

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orangehorse801
7 Feb 2017
School
University of Windsor
Department
Biological Sciences
Course
BIOL 3581
Professor
Vacratis

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Document Summary

Schematic figure of cell culture week: only the first week. When you use electricity to transfer your proteins onto the inert membrane, it gives. Immunoblotting is more of a scientific name you a replica of what the gel looked like. Coomassie blotting allows you to see total protein. But you are interested in looking at and detecting a protein of interest. There are a variety of different ways to do quantitiative expression analysis, pull down protein complex (pull down one and use an antibody to detect another one to see if it came down with it) A variety of different applications but the goal of antibody use is to be able to use an antibody that is specific for a protein in order to make some sort of conclusion based on it. In the electroblotting technique, you get this exact replica of what was on the gel.

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Related Questions

A student written question: New Belgium Brewery creates its own yeast strains for brewing beer. Some yeast strains secret a toxic protein, which is not detectable until after the brewing process. Therefore the brewery must find a way to detect the yeast that secretes this toxic protein. Because they collaborate with Dr. Wolkow, a yeast-whiz, the brewery decides to hire you to work with Dr. Wolkow to make a cDNA expression library from the toxin-secreting strain. You decide to use a bacterial expression vector since it is easy to work with.

1. What components must the expression vector contain?

a. promoter, translation start sequence, cloned cDNA sequence, DNA polymerase

b. DNA ligase, translation start signal, cloned cDNA sequence, polyA tail

c. Shine-Dalgarno sequence, promoter, cloned cDNA sequence, antibody

d. promoter, Shine-Dalgarno sequence, cloned cDNA sequence, transcription termination sequence

2. Suppose the radioactive 3 antibody did not attach to any of the protein products. What can you conclude from this?

a. the toxic protein product is produced by all the yeast strains

b. the toxic protein product is produced by one or more of the yeast strains

c. the toxic protein product is not produced by any of the yeast

d. the yeast DNA was destroyed when the cells were lysed

3. Why would the brewer want to specifically use an expression vector rather than another type of vector?

a. It is specifically for eukaryotic DNA

b. It is used to produce the protein encoded by a cloned gene

c. It is only used for cloning cDNA sequences

d. They want to produce mRNA encoded by a cloned gene

4. This type of blot, where protein is transferred onto the membrane and is then hybridized to a tagged-antibody is a blot.

a. Southern

b. Northern

c. western

d. southwestern

Which of the following did Erwin Chargaff observe?

A)

Base composition changes as the organism ages.

B)

Base composition of DNA does not vary from one species toanother.

C)

In all species the number of adenines equals the number ofguanines.

D)

In all species A + T = G + C.

E)

DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)

the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)

the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)

the temperature at which double-stranded DNA is completelymelted.

D)

the temperature at which double-stranded DNA has become 50%single-stranded.

E)

the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)

Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)

Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)

Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)

A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)

Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)

Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)

Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)

When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)

None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)

DNA ligase

B)

Restriction endonuclease

C)

Reverse transcriptase

D)

DNA polymerase I

E)

Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)

Reverse transcriptase

B)

Alkaline phosphatase

C)

DNA ligase

D)

DNA polymerase I

E)

Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)

Alkaline phosphatase

B)

Reverse transcriptase

C)

Restriction endonuclease

D)

DNA ligase

E)

DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)

An origin of replication

B)

A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)

Unique restriction sites for insertion of DNA.

D)

Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)

All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)

Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)

cDNA libraries contain cDNAs made from mRNA.

C)

Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)

Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)

cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

A)

dNTPs

B)

DNA containing the sequences to be amplified

C)

DNA ligase to connect the fragments together

D)

Primers complementary to each end of the sequence to beamplified

E)

Heat stable Taq polymerase

How can a fusion protein be formed?

A)

Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)

Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)

Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)

Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)

A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)

The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)

The reaction is only dependent on the presence of GFP and oneother cofactor.

C)

It is easily cloned and expressed in bacterial cells.

D)

The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)

It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)

Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)

Create a fusion with green fluorescent protein (GFP).

C)

Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)

A and B might work.

E)

A, B, and C might work.

F)

None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)

The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)

A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)

Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)

Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)

The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)

Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)

Eukaryotic genes often encode introns that are spliced outbefore translation.

C)

Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)

Eukaryotic genes are typically arranged in operons.

E)

Prokaryotic genes often encode exons that are spliced out beforetranslation

1.

__________ are fairly small organelles that provide a safe place within the cell to carry out certain biochemical reactions that generate harmful, highly reactive oxygen species. These chemicals are both generated and broken down in the same location.

Lysosomes
Endosomes
Peroxisomes

Nucleosomes

2.

Biologists cannot possibly study all living species. Instead, they try to understand cell behavior by studying a select subset of them. Which of the following characteristics are useful in an organism chosen for use as a model in laboratory studies?

rapid rate of reproduction
all of the above
ability to grow under controlled conditions

amenability to genetic manipulation

3.

By definition, prokaryotic cells do not possess __________.

ribosomes.
a nucleus.
membrane bilayers.

replication machinery.

4.

Cell biologists employ targeted fluorescent dyes or modified fluorescent proteins in both standard fluorescence microscopy and confocal microscopy to observe specific details in the cell. Even though fluorescence permits better visualization, the resolving power is essentially the same as that of a standard light microscope because the resolving power of a microscope is limited by the __________ of light.

intensity
absorption
wavelength

filtering

5.

Choose the phrase that best completes this sentence: Microtubules ____________ and are required to pull duplicated chromosomes to opposite poles of dividing cells.

generate contractile forces
can rapidly reorganize
are found in especially large numbers in muscle cells

are intermediate in thickness

6.

Despite the differences between eukaryotic and prokaryotic cells, prokaryotes have proteins that are distantly related to eukaryotic actin filaments and microtubules. What is likely to be the most ancient function of the cytoskeleton?

cell division
cell motility
vesicle transport

membrane support

7.

DNA and RNA are different types of nucleic acid polymer. Which of the following is true of DNA but not true of RNA?

It contains thymine.
It has 5'-to-3' directionality.
It is single-stranded.

It contains uracil.

8.

Double covalent bonds are both shorter and stronger than single covalent bonds, but they also limit the geometry of the molecule because they ____________________.

prevent additional bonds from being formed with the bonded atoms.
limit the rotation of the bonded atoms.
create a new arrangement of electron shells.

change the reactivity of the bonded atoms.

9.

Drosophila melanogaster is a/an __________. This type of animal is the most abundant of all animal species, making it an appropriate choice as an experimental model.

mammal
amphibian
bird

insect

10.

If the isotope 32S has 16 protons and 16 neutrons, how many protons, neutrons, and electrons will the isotope 35S have, respectively?

16, 19, 17
16, 19, 15
16, 19, 16

16, 20, 15

11.

Living systems are incredibly diverse in size, shape, environment, and behavior. It is estimated that there are between 10 million and 100 million different species. Despite this wide variety of organisms, it remains difficult to define what it means to say something is alive. Which of the following can be described as the smallest living unit?

cell
organelle
protein

DNA

12.

Macromolecules in the cell can often interact transiently as a result of noncovalent interactions. These weak interactions also produce stable, highly specific interactions between molecules. Which of the factors below is the most significant in determining whether the interaction will be transient or stable?

the concentration of each molecule
the size of each molecule
surface complementarity between molecules

the rate of synthesis

13.

Many of the mechanisms that cells use for maintenance and reproduction were first studied at the molecular level in bacteria. Which bacterial species had a central role in advancing the field of molecular biology?

E. coli
S. pombe
C. elegans

D. melanogaster

14.

Many types of cells have stores of lipids in their cytoplasm, usually seen as fat droplets. What is the lipid most commonly found in these droplets?

cholesterol
isoprene
triacylglycerol

palmitic acid

15.

Mitochondria contain their own genome, are able to duplicate, and actually divide on a different time line from the rest of the cell. Nevertheless, mitochondria cannot function for long when isolated from the cell because they are __________________.

anaerobes.
viruses.
parasites.

endosymbionts.

16.

Mitochondria perform cellular respiration, a process that uses oxygen, generates carbon dioxide, and produces chemical energy for the cell. Which answer below indicates a correct pairing of material “burned” and the form of energy produced during cellular respiration?

fat, protein
sugar, fat
sugar, ATP
fat, ADP

17.

Polar covalent bonds are formed when the electrons in the bond are not shared equally between the two nuclei. Which one of these molecules contains polar bonds?

propane
methane
molecular oxygen
water