Bridging Exercise Gene Transfer, Recombinant DNA and Genetic Engineering

Explain 2 methods of artificial transformation

(competency has to be induced) 1. Calcium Chloride - Ca2+ neutralizes - charge in cell envelope, - charged DNA can move across , then cold shocked, then heat shocked, the heat on the outside of the cell facilitates the movement of the plasmid into the cold interior of the cell (cold then hot increases fluidity of membrane) 2. Liposome - DNA coated with lipid, fusion of lipid with the membrane, facilitating entry of DNA

Percentage of bacteria that are capable of natural transformation in lab

1%

What are the 3 possible results from Blue White screening?

1) Bacteria are not transformed (no plasmid) → they don't have the amp resistance gene → they can't grow on amp media. 2) Bacteria are transformed (they pick up plasmid, but not the recombinant plasmid) → they have amp resistance gene -> they can grow on amp media, but their lac operon was not interrupted so they produce galactosidase (lactase) → hydrolyzes chromogenic galactoside (lactose) → blue color 3) Bacteria are transformed (they pick up recombinant plasmid) - they have amp resistance gene -> they can grow on amp media and their lac operon was interrupted so they cannot produce galactosidase (lactase) → NO hydrolysis of chromogenic galactoside (lactose) → white color

Explain the process of the Southern blot method.

1) Restriction endonucleases are used to cut DNA strands into smaller fragments called RFLP's. 2) RFLP's are then electrophoresed on an agarose gel to separate them by size. 3) Treat agarose gel with NaOH to denature double stranded DNA (separate into single stranded DNA - necessary so that single stranded DNA probe can bind) 4) A sheet of nitrocellulose membrane is placed on the gel to transfer to the DNA to the membrane (DNA is negatively charged, membrane is positively charged). 5) The membrane is exposed to a hybridization probe—a single stranded DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labeled with a chromogenic, fluorescent, or radioactive tag. 6) After hybridization, excess probe is washed from the membrane and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive probe, a UV light in the case of a fluorescent probe, or by development of color on the membrane if a chromogenic probe.

20 cycles of PCR result in how many DNA molecules? (assume you start with 1 molecule)

1,048,576

What are 2 commonly used marker genes?

1. Ampicillin resistance gene (beta lactamase gene) - makes bacteria resistant to beta-lactam antibiotics like penicillins; so only bacteria that have taken up the plasmid with this gene will be able to grow on media that contains ampicillin 2. Beta-galactosidase (lacZ) gene in the lac operon - the gene of interest is inserted into the lac operon, interrupting the structural gene that codes for the enzyme beta-galactosidase.

Two major goals of genetic engineering using recombinant DNA tech

1. Amplification 2. Protein synthesis

Briefly explain the 3 steps of the PCR method

1. Denaturation: Heat the DNA to near boiling; this denatures the DNA (splits the double stranded DNA into two single strands). 2. Annealing: Cool down and add "forwards" and "backwards" primers (short strands of DNA); primers complementary base pair to single strands of DNA. 3. Extension: Warm up and add free nucleotides and DNA polymerase III to finish replication.

Explain 3 physical methods of artificial transformation

1. Electroporation - brief shock cells which forms pores in membrane. Plasmid DNA can enter the cell, natural mech will rapidly close pores after shock 2. Microinjection - into animal cells using glass pipette 3. Biolistic particle delivery - paricles of a heavy metal coated with plasmid DNA the particles are then shot into cells with a burst of helium 4. Temp shock - first cells are incubated on ice and then in a warm water bath. Heat creates a temp gradient and DNA pulled into cell

What are 2 ways scientists obtain genes they are interested in?

1. Genomic libraries - genome is treated with restriction endonucleases, then RFLP's are inserted into "books" (strains of bacteria, yeast, or bacteriophages). 2. Synthetic DNA - DNA synthesis machines can synthesize short sequences of DNA (up to ~120 nucleotides long); sequence must be known before it can be synthesized

Why is Bacillus subtilis often preferred over E. coli for making gene product? (2 reasons)

1. It's G(+), so has no outer membrane and no lipopolysaccharides (LPS) that can act as endotoxins (gene product or protein can then be contaminated with endotoxins) 2. It's more likely to secrete its gene product (protein); E. coli must be lysed to isolate the gene product (protein) and lipopolysaccharides from the outer membrane may contaminate the gene product

Identify 2 general methods for identifying transformed bacteria.

1. Marker genes - include in plasmid 2.Use radioactive or fluorescent probes that will complementary base pair with the gene of interest

What are 3 properties of a good plasmid vector?

1. Small size (easier manipulation) 2. Circular shape (protects against restriction enzymes) 3. Self replicating (has origin of replication)

Explain the process of insulin production using recombinant DNA technology

1. synthetic genes are first constructed for each of the two short polypeptide chains that make up the protein 2. each gene is inserted into a plasmid vector and linked to the end of the marker gene coding for the bacterial enzyme beta-galactosidase (hormone is coproduced with enzyme) 3. polypeptides are recovered, separated from the enzyme, and chemically joined to make insulin.

Explain Blue-White Screening

2 markers are included in the plasmid: one for antibiotic resistance and one for the production of the enzyme beta-galactosidase (lacZ) used in the hydrolysis of galactosides (lactose). The gene of interest is inserted into the lacZ gene to create a recombinant plasmid; this insertion interrupts the lacZ gene so that galactosidases cannot be produced. Bacteria are transformed and are then grown on media containing ampicillin and galactosides (a special form of lactose is use - when lactose is hydrolyzed, one of the products is chromogenic - it's blue!). Only bacteria that are ampicillin resistant will grow on this media (meaning they picked up the plasmid). Now to determine if they picked up the recombinant plasmid: If the bacteria pick up the recombinant plasmid, they will NOT be able to hydrolyze lactose and will produce white colonies. If the bacteria received the original plasmid containing the intact lacZ gene, the cells will hydrolyze lactose to produce the blue-colored compound, making the colony blue.

What does Bt stand for?

Bacillus thuringiensis (species of bacteria)

What does heat do to a double stranded nucleic acid molecule like DNA?

Breaks double bonds between paired nitrogenous bases, separating the 2 DNA strands.

Lysogenic virus examples

Chicken pox, Human papilloma virus, HIV, and Herpes

c in cDNA stands for

Complementary

The transfer of DNA from one bacterial cell to another by pilli

Conjugation

Why can't eukaryotic DNA be put directly into prokaryotic chromosome?

Contains introns

cDNA and Reverse transcription sequence of events

DNA premRNA mRNA mRNA-cDNA Denature: sscDNA and mRNA DS cDNA

F(+) cells do or do not have pilli

DO

What is this RNA's role in gene silencing and RNA interference? (Explain how it works against an RNA virus)

DS siRNA is synthesized and then denatured to produces 2 single strands. One strand is degraded. The strand that is kept is complementary to the viral RNA. So when mixed with viral RNA, the SS siRNA binds to the viral RNA and viral replication is halted.

What was accomplished in the Human Genome Project?

Entire human genome was sequenced.

What does F in F plasmid stand for

Fertility

Why isn't bacterial DNA harmed by restriction enzymes

Foreign DNA that is not methylated are degraded by restriction enzymes

Bioinformatics is the field of DNA sequencing using databases called

GenBank

A fragment of DNA will be inserted into a viral capsid, and the virus then infect another cell (virus a vector for genetic material from one bacterium to another)

Generalized transduction

Give 2 examples of this kind of vaccine

Hepatitis B, Gardasil (against HPV), acellular Pertussis

What will be accomplished by the Human Proteome Project?

Identify proteins encoded in genes

Name 2 hormones produced by recombinant DNA techniques

Insulin & human growth hormone

DS cDNA can be inserted into bacterial chromosome because it lacks

Introns

Noncoding sequences of DNA

Introns

What is the advantage of using a fungus over a prokaryotic organism?

It's eukaryotic (don't have problems with exons and introns) and it's more likely to secrete its gene product (protein)

Infection of a cell which a virus becomes latent by becoming integrated a host's cells chromosome is called the

Lysogenic cycle

What is an advantage of the using this kind of vaccine? (subunit)

No risk of acquiring the pathogen/disease from the vaccine.

What is a common method of DNA amplification?

PCR (polymerase chain reaction)

For DNA fingerprinting, what usually must be done to the DNA before using the Southern Blot method?

PCR to amplify the DNA

cDNA made from process called

REVERSE TRANSCRIPTION - DNA produced from mRNA

What does RNAi stand for?

RNA interference

DNA that contains genes from more than one organism is called

Recombinant DNA

What does the R in R plasmid stand for

Resistance

Enzymes synthesized by bacterial cells to cut apart and destroy viral DNA

Restriction endonuclease

What does RFLP stand for?

Restriction fragment length polymorphism

Viral family gave the idea from reverse trancriptase enzyme

Retroviridae. Retroviruses - HIV

Enzyme the catalyzes reverse transcription to yield cDNA

Reverse transcripase

Enzyme that makes mRNA from preRNA

Ribozymes - excise introns, splice exons together

What is golden rice?

Rice that has been genetically engineered to produce vitamin A

A common technique for detecting a gene of interest is the:

Southern Blot method.

A prophage improperly exits from bacterial chromosome, takes adjacent bacterial DNA with it and infects other bacterial cell

Specialized transduction

Why is it possible to use synthetic genes in the production of insulin?

The 2 polypeptide chairs are only 21 and 30 amino acids long; because they are short, they can be synthesized in the lab (the max that can be synthesized is about 120 nucleotides).

Which of the following would represent the ampicillin resistant cells? The ampicillin sensitive cells? (Use an R & an S)

The cells that didn't grow S The cells that grew and are blue R The cells that grew and are white R

Which of the following would represent the cells that have been transformed by the recombinant plasmid (the desired effect)?

The cells that grew and are white

What is the purpose of treating RFLP's with NaOH in the Southern Blot procedure?

To denature double stranded DNA (separate into single stranded DNA - necessary so that single stranded DNA probe can bind)

A genetic change in which DNA leaves one bacterial cell, exists for a time in the aqueous ECF and then is taken into another cell the it may be incorporated into chromosome

Transformation

DNA molecule used as a vehicle to transfer foreign material into another cell

Vector

2 common vectors

Viruses(bacteriophages) and plasmids

A virus that infects a bacteria is a

bacteriophage

What do we call this process?

denaturation

cDNA is complementary to

mRNA (acts as template and complementary base pairing occurs to build cDNA)

DNA amplification

making multiple copies of DNA

A viral nucleic acid that becomes integrated in the bacterium's chromosome called a

prophage (provirus if in eukaryote)

Noncoding sequences, once called junk DNA, includes:

regulatory sequences, codes for RNA (tRNA & rRNA), telomeres, introns, etc.

What enzymes are used to produce RFLP's?

restriction endonucleases

Gel electrophoresis is used to separate RFLP's by _______________.The longer the fragment, the _______ the distance it travels in the gel.

size (molecular weight), shorter

What does siRNA stand for?

small interfering RNA

Restriction endonuclease produce staggered cuts called

sticky ends

A _______ vaccine is one that consists only of the antigenic protein portion of a pathogen

subunit vaccine

Give specific examples of genetically engineered crops.

• Bt as an insecticide: Crops can also be genetically modified to produce this Bt toxin; insects die when they eat the plant. • Crop resistance to Roundup (herbicide) • Golden rice - genetically engineered to produce vitamin A; • Rye has been genetically engineered to resist infection with the fungus Claviceps purpurea, which produces ergot toxin.