molecular genetics exam 1

A researcher proposes the use of gene cloning to cure the genetic disease sickle cell anemia. This treatment would include inserting a copy of the Hemoglobin-A gene into a plasmid vector, expressing the protein in E. coli cells and purifying the protein, which could then be injected into patients with sickle cell anemia. Do you expect this therapy to be successful in treating patients with sickle cell anemia?

no bc The hemoglobin beta protein purified from E. coli cells will not contribute to the structure of existing red blood cells.

DNA replication

The biological process of making an identical copy of DNA, which occurs each time a new cell is formed in living organisms. The double helix is unwound, and each strand of the original molecule serves as a template for the production of the complementary strand.

In genetic engineering, plasmid vectors are often used to express a protein of interest in a cell. While the protein expressed and other features of the plasmid can vary, all plasmid vectors must contain certain common elements that are critical to their function. Which of the following components of plasmids are necessary to make them ideal vectors for genetic engineering?

ori site promoter antibiotic resistance gene

gene

the fundamental physical and functional unit of heredity; an ordered sequence of nucleotides located in a specific place on the DNA that encode for a specific functional product

gel electrophoresis

the movement of charged molecules toward an electrode of the opposite charge; used to separate nucleic acids and proteins. When used to separate DNA fragments, electrophoresis will separate the fragments by size, with smaller fragments moving faster than larger fragments

genetic engineering

the process of altering the genetic material of cells or organisms to enable them to make new substances or perform new functions

restriction enzyme sites

these are regions that are cut by enzymes allowing for segments to be inserted

antibiotic resistance gene

this codes for a protein that inactivates a target antibiotic -- also this allows you to identify which bacteria has your vector plasmid

Digestion

The cutting of DNA by a restriction enzyme

Genetic engineering of bacterial cells can result in production of a human protein of interest by which of the following processes:

The gene of interest is inserted into a bacterial cell that can complete transcription and translation of that gene

Which of the following factors can impact protein folding?

The pH of the buffer a protein is stored in. The environmental temperature. The hydrophobic or hydrophilic nature of the amino acids that make up a protein.

Transcription

The process by which information encoded in DNA is transferred to messenger RNA, a single-stranded ribonucleic acid.

Antibiotic resistance:

The state in which bacteria are no longer sensitive to an antibiotic and will continue to grow and divide in the presence of the antibiotic.

loading dye in gel electrophoresis

this is added to the well so the sample can sink the dye consist of: glycerol, dense liquid (the dye also shows that the sample is loaded, running in correct direction, and distance traveled after gel electrophoresis

origin of replication

this is where the proteins that initiate replication binds

promoter

this region where RNA polymerase binds to start transcription

Base pair

Two complementary nitrogen-containing molecules paired together in double-stranded DNA by weak bonds.

Plasmid

a circular molecule of DNA

genetic disease

a disease caused by a change in DNA. Genetic diseases are often inherited from parents

diabetes

a disease that occurs when the body doesn't produce or properly use insulin

DNA (deoxyribonucleic acid)

a double stranded molecule made up of nucleotide subunits that encodes genetic information

Insulin

a hormone produced in the pancreas that controls the amount of glucose in the blood. Insulin is a protein.

protein

a large biomolecule. proteins carry out essential functions in cells, from forming cellular structures to enabling chemical reactions to take lace. examples of proteins are enzymes, red fluorescent protein, cell receptors, and some hormones

Nucleotide

a set of molecules that link together to form DNA or RNA. DNA and RNA each contain four types of nucleotides.

sequence

a set of related events, movements, or things that follow each other in a particular order.

Which features from the pKAN-R and pARA plasmids should be included in your new recombinant plasmid to produce RFP in E. coli cells? Select all that apply.

antibiotic resistance gene (either AmpR or KanR) gene for red fluorescent protein expression (rfp) pBAD promoter origin of replication (ori) araC gene that regulates the pBAD promoter correct

what operon was discovered in E.Coli and is often modified for use in genetic engineering applications?

araBAD operon ( controls metabolism of sugar arabinose)

what is the best pH and temperature for a restriction enzyme?

37 C and pH of 7

biomolecule

A carbon based molecule made by living things. most are long polymers mad of repeating subunits called monomers- and include proteins, CHO, lipids, and nucleic acids

Plasmid

A circular molecule of DNA.

Antibiotic:

A class of compounds that kill or inhibit the growth of microorganisms.

Hemophilia

A disease that occurs when the ability of blood to clot is reduced due to lack of one or more blood clotting factors.

Growth deficiency

A disease that occurs when the body doesn't produce enough human growth hormone.

Arabinose:

A five-carbon sugar that naturally occurs in various plant and bacterial carbohydrates.

Selectable marker:

A gene on a plasmid that is introduced into a cell along with a gene of interest that is being cloned. Selectable markers allow scientists to tell if the plasmid has been taken in by the cell because the marker can be seen or detected. A common selectable marker is an antibiotic resistance gene—only bacteria that have the gene will survive the antibiotic.

human growth hormone

A hormone secreted by the pituitary gland that stimulates growth. Human growth hormone is a protein.

Micropipette

A laboratory instrument used to measure, dispense, and transfer very small amounts of liquid.

Supercoiled

A plasmid configuration consisting of a single plasmid that has been twisted. The shape of this plasmid is more compact (takes up less space) than the circular form.

Multimer

A plasmid configuration consisting of multiple plasmids that have interlocked during formation so they are like links in a chain.

Nicked circle

A plasmid configuration that consists of a single plasmid that has a break in one of its two strands. The shape of this plasmid is circular

Which statement best describes a plasmid?

A plasmid is a piece of circular DNA present in bacterial cells that carries genes that confer new traits to a bacterial cell.

agarose

A polymer made up of sugar molecules that is used as the matrix in gel electrophoresis procedures.

Bacterial conjugation

A process by which two bacterial cells join and transfer genetic material from one to another.

Enzyme

A protein that catalyzes a chemical reaction

RNA polymerase

A protein that makes messenger RNA from DNA.

Activator:

A protein that regulates transcription of a gene by binding to a sequence near the promoter, thus enabling RNA polymerase to bind to the promoter and initiate transcription of the gene. The activator protein can also block the binding of RNA polymerase and thereby inhibit transcription of the gene.

What is a replication fork?

A replication fork is a point in a DNA molecule where the two strands separate during replication.

Origin of replication (ori)

A sequence of DNA at which replication of the DNA is initiated.

Loading dye

A set of dyes that are added to biomolecules such as DNA for gel electrophoresis. One dye moves farther than the sample, which indicates that it is time to stop running the gel.

RNA (ribonucleic acid)

A single-stranded biomolecule made up of a nitrogenous base, a ribose sugar, and a phosphate; RNA plays a critical role in protein synthesis, transmitting genetic information from DNA to the ribosome where proteins are then made.

The cloning of a DNA fragment requires connecting a gene to a plasmid after both have been digested with the same restriction enzyme. Name the enzyme that connects DNA and plasmid fragments together:?

ligase

amp R codes for what?

B lactamase which inactivates ampicillin and allows for growth

Which restriction enzyme(s) should you use to create a recombinant plasmid from the pKAN-R and pARA plasmids shown above, such that the plasmid can be maintained in a bacterial population and produce RFP?

BamHI and HindIII

Why does gel electrophoresis separate genetic material into bands?

Because it conducts a current that causes the DNA material to move by its charge and size.

Why is it necessary to verify a recombinant plasmid?

Because multiple recombinant plasmids are possible, and you want to make sure that the expected plasmid was successfully generated.

How does altering an organism's DNA change its traits?

Changing the DNA alters the proteins that are produced, which are responsible for the traits exhibited by an organism.

Choose the strongest interaction within a double-stranded DNA molecule.

Covalent bonds between adjacent nucleotides

A plasmid is an extrachromosomal element composed of _________ and _________.

DNA & double stranded

Recombinant DNA

DNA that contains sequences or genes from two or more sources.

Sticky ends

Ends of a DNA molecule cut with certain restriction enzymes. These ends are asymmetrical in that one strand is longer than the other strand and therefore has unpaired bases. The sticky ends of two different pieces of DNA that have been cut with the same restriction enzyme(s) can be joined, as the unpaired bases on their ends are complementary.

Which of the following functions can the pARA plasmid carry out when present in bacterial cells?

Initiate replication of the pARA plasmid. - Produce a protein that allows bacteria to survive in the presence of that antibiotic Ampicillin.

Which of the following functions can the pKAN-R plasmid carry out when present in bacterial cells?

Initiate transcription of the rfp gene. -Produce a protein that allows bacteria to survive in the presence of that antibiotic Kanamycin.

What role did DNA ligase play in producing the recombinant plasmids you are verifying?

It joins complementary DNA fragments together.

How does genetic engineering work?

Normally insulin is produced in the pancreas, but in people with type 1 diabetes there is a problem with insulin production. People with diabetes therefore have to inject insulin to control their blood sugar levels. Genetic engineering has been used to produce a type of insulin, very similar to our own, from yeast and bacteria like E. coli. This genetically modified insulin, 'Humulin' was licensed for human use in 1982.

5. What is the relationship between proteins and traits?

Proteins produced by expression of genes result in traits.

The genetic engineering process

A small piece of circular DNA called a plasmid is extracted from the bacteria or yeast cell. A small section is then cut out of the circular plasmid by restriction enzymes, 'molecular scissors'. The gene for human insulin is inserted into the gap in the plasmid. This plasmid is now genetically modified. The genetically modified plasmid is introduced into a new bacteria or yeast cell. This cell then divides rapidly and starts making insulin. To create large amounts of the cells, the genetically modified bacteria or yeast are grown in large fermentation vessels that contain all the nutrients they need. The more the cells divide, the more insulin is produced. When fermentation is complete, the mixture is filtered to release the insulin. The insulin is then purified and packaged into bottles and insulin pens for distribution to patients with diabetes.

Promoter

A specific DNA sequence that binds RNA polymerase and initiates transcription of the gene.

Recognition site

A specific DNA sequence that is cut by a restriction enzyme. Many restriction sites are palindromes, sequences that are the same when read forward or backward.

restriction site

A specific sequence on a DNA strand that is recognized and cut by a restriction enzyme. they are 6-8 bases long often palindromic

Restriction digest:

A technique in which naturally occurring enzymes are used to cleave DNA at specific sequences.

blood clotting factor

A variety of proteins in blood plasma that participate in the clotting process.

Vector

A vehicle for moving DNA sequences from one organism to another.

Bacteriophage

A virus that infects a bacterial cell and uses the cell machinery to replicate itself, eventually destroying the bacterial cell.

identify the processes below that are examples of genetic engineering. Select all that apply.

A)Expressing the human gene for insulin in bacterial cells to produce insulin for diabetes patients. B)Creating a rice plant that produces beta carotene to provide a key nutrient lacking from regional diets. C)Growing a new organism, like the famous sheep Dolly, by removing the DNA of a reproductive cell and replacing it with the DNA from a different cell. D)Using techniques such as CRISPR to alter the DNA sequence of mosquito genes so that a population becomes resistant to malaria.

Messenger RNA

An RNA molecule transcribed from the DNA of a gene and used as the template for protein synthesis.

Restriciton Enzyme

An endonuclease (type of enzyme) that recognizes and cuts DNA molecules foreign to a bacterium (such as phage genomes). The enzyme cuts at specific nucleotide sequences (restriction sites).

DNA ligase

An enzyme that catalyzes the formation of covalent chemical bonds in the sugar-phosphate backbone, thereby binding fragments of DNA together.

Why are the cells incubated at 42°C?

bacteria begins to degrade and create pores where dna now can enter the cells

how do you prevent restriction enzymes from cutting their own DNA?

bacteria use a methylase enzyme to add a methyl group to their own DNA to protect it

cell

basic unit of any living organism that carries on the biochemical process of life

Consider a circular DNA plasmid that has been cleaved (cut) once a by a restriction enzyme. Which type of cleavage would be the most difficult to reconnect?

blunt ends

what type of cut does smal do?

blunt ends no overhanging

how do you determine if the bacteria has the plasmid with the GOI in the agar plate after growing?

by having a antibiotic resistance gene in the plasmid.. when you grow on plate you add the nutrients so it can grow and also a antibiotic ... so when colony grows you know they have your GOI in plasmid since it grew on the agar when only colonies resistant will grow

what is The Sanger sequencing method, developed in 1977,

enabled scientists to read the genetic code for the first time. It is based on the natural process of DNA replication

what breaks hydrogen bonds in a double stranded DNA that creates the replication bubble?

helicase( a enzyme)

Complementary base pairs in the DNA double helix are held together by what type of interaction?

hydrogen bonds

what happens to DNA when it is heated at high temperatures?

it becomes unstable, it starts to denature, and also involves the breaking of hydrogen bonds that bind complementary

Why is ampicillin added to the agar?

it only allows bacteria to grow that are resistant , also indicating transformation was a success

What is DNA polymerase III?

it reads the DNA and creates a new copy, it reads from 3' to 5' traveling away from replication fork. creating the new DNA in 5' to 3' .... traveling away from 3' is called the leading strand

You have a piece of DNA containing the gene for green fluorescent protein (gfp) and the pBAD promoter. This DNA fragment has been cleaved with BamHI and HindIII and can be inserted into either the pKAN-R plasmid or the pARA plasmid. Which plasmid would you choose to use as the plasmid vector to express gfp protein in bacterial cells? explain your answer

pARA plasmid 1)The pKAN-R recombinant plasmid would not be maintained in bacterial cells as they replicate. 2)Once the gfp DNA fragment is inserted, the pARA plasmid could express the gfp protein. 3)The pARA recombinant plasmid would be maintained in bacterial cells as they replicate.

What is bacterial transformation?

process by which bacterial cells take up naked DNA molecules

how does a prokaryotic cell is altered to allow binary fission to occur

smaller lack organelles and complex replication machinery prokaryotic fxn faster rate than comparable eukaryotic enzymes making binary fission and other bacterial processes fast and efficient, allowing bacteria to respond rapidly to their environment

What is the DNA Ladder (M) composed of and why do you need it?

standard mix of DNA fragments of known length, used to estimate the size of unknown fragments

what type of cut does Ecori do?

sticky ends overhanging