Mastering Genetics HW18

You have isolated a plasmid from E. coli. You get the following results, using three restriction enzymes, 1) XhoI, 2) BamHI, and 3) EcoRI to perform six different digestions: single digests using each enzyme alone and double digests using each combination of two enzymes. enzyme #fragments size (in kb) XhoI 1 10 BamHI 2 3.75, 6.25 EcoRI 2 4.25, 5.75 XhoI+BamHI 3 1.25, 3,75, 5 XhoI+EcoRI 3 2, 3.75, 4.25 BamHI+EcoRI 4 1.25, 2.5, 3, 3.25 Drag the appropriate labels to their respective targets.

(start from top EcoRI and continue clockwise) EcoRI --> 2.5 kb --> BamHI --> 1.25 kb --> XhoI --> 2 kb --> EcoRI --> 3 kb --> BamHI --> 1.25 kb --> back to first EcoRI

You ligate a gene into the plasmid shown in Part A, transform the plasmid into bacteria, and plate the cells on three types of media: a) complete media, b) complete media + ampicillin, and c) complete media + ampicillin + X-gal. Complete the tables by answering each question about the colonies of bacteria that grow on each type of media. 1. What color would you expect the colonies to be? (white only, blue only, white and blue) 2. Without additional experimentation, could you predict whether the cells in the colonies contain the plasmid? (yes or no) 3. Without additional experimentation, could you predict whether the cells in the colonies contain the gene of interest? (yes or no)

1. a) white only b) white only c) white and blue 2. a) no b) yes c) yes 3. a) no b) no c) yes The ampR gene produces a protein that breaks down ampicillin. Only cells that contain the plasmid with the ampR gene will be able to grow on plates that contain ampicillin. The lacZ gene encodes a protein that produces a blue color in the presence of X-gal. The multiple coding site (MCS) for the plasmid is located within the lacZ gene. Therefore, if a DNA fragment is ligated into the MCS, the lacZ gene will no longer function and will not produce the blue color. In the presence of X-gal, cells that contain a plasmid with a fragment inserted into the MCS will be white, whereas cells that contain a plasmid without a fragment inserted into the MCS will be blue.

What size fragments you would expect from an EcoRI/XhoI double digest of the plasmid below?

2 kb, 3.75 kb, 4.25 kb

Half of the palindromic restriction site that the endonuclease PstI recognizes, binds, and cleaves is 5'-CTG_____-3'. What is the complete sequence?

5'-CTGCAG-3'

The human genome is 3 * 10^9 bp in length. Calculate how many fragments would result from the complete digestion of the human genome with EcoRI (5'-G (down arrow) AAATTC-3').

7.32 * 10^5 6bp sequence recognition enzyme (1/4)^6 * (3 * 10^9) = 732,422 fragments = 7.32 * 10^5

A single nucleotide in a genome can be targeted for mutagenesis using the ________ technique, adapted from a bacterial defense system.

CRISPR-Cas9

Which method could be used to delete a mutant exon of the dystrophin gene in muscle cells as was demonstrated by genetic therapy for mice with Duchenne muscular dystrophy?

CRISPR-Cas9 genome editing

Which of the following best lists the correct order of the steps taken to produce human insulin in E.coli in the 1970s?

III, IV, I, II

Which of the following is a possible advantage for the environment of having farmers use plants that genetically produce Bt toxin?

Plants with genes for the toxin are eaten less often by insects, and therefore less insecticide is needed.

In producing a recombinant plasmid to be used to clone a given donor insert, it is possible to cut both the donor and plasmid with the same restriction enzyme, resulting in complementary sticky ends. Assuming plenty of plasmid DNA is available, why is further selection necessary before the introduction of the plasmid into a cellular system?

Some donor strands will be inserted with an incorrect orientation.

In using Agrobacterium tumefaciens to transfer genes into plants, what is transferred from bacterium to plant?

T-DNA

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan hound, Tai, into the enucleated egg from a donor. The embryo was transplanted into a surrogate mother to develop to term. Which of the following animals would be a genetic match for Snuppy if *nuclear* DNA was tested?

Tai

In selecting recombinant bacteria, cells are chosen that are resistant to a specific antibiotic. How are the bacteria made resistant?

The antibiotic resistance gene is encoded in the vector.

Which of the following statements is FALSE regarding gene therapy?

The integration of a trans gene into the genome of the target cell by a viral vector is not permanent, and therefore it will require repeated treatments.

If bacteria transformed with a recombinant pUC18 plasmid produce white colonies when grown on plates containing ampicillin and Xgal, which of the following is least likely?

They product functional beta-galactosidase that cleaves X-gal. *true responses:* - They produce Beta-lactamase that provides resistance to ampicillin. - They carry a vector with a lacZ gene that has been disrupted and rendered nonfunctional. - They carry a vector that contains a DNA fragment inserted into the multiple cloning site. - They carry a vector with a bla gene used as a selectable marker.

The binary approach uses both a transformation vector that contains the T-region flanked by border sequences and a disarmed ___________ with the genes required for virulence and conjugative transfer.

Ti plasmid

Which of the following statements regarding restriction enzymes is NOT true? a) Most restriction enzymes recognize sequences of 20-25 bp in length. b) Bacteria can add methyl groups in their DNA to prevent digestion by endogenous restriction enzymes. c) Most restriction enzymes recognize palindromic sequences in host DNA. d) Restriction enzymes may produce either sticky or blunt ends when digesting host DNA.

a) Most restriction enzymes recognize sequences of 20-25 bp in length. Most restriction enzymes recognize palindromic sequences of 4-8 bp in length.

Below is an image of a typical cloning plasmid. Match each element in the plasmid with its most important function in cloning. Boxes by a) ampicillin resistance gene (ampR) b) DNA sequencing primer site c) Multiple cloning site d) lacZ gene e) origin of replication (ori) [notes from mastering genetics]

a) allows for selection of bacterial cells that contain the cloning plasmid [- allows for selection of cells that contain a cloned DNA fragment] b) allows for identification of the DNA segment cloned into the plasmid [allow for the cloned DNA to be sequenced and identified] c) region where pieces of DNA are cloned into the plasmid [region that contains multiple restriction enzyme cut sites that are used to clone the DNA of interest] d) allows for selection of bacterial cells that contain a cloned DNA fragment e) enables replication of the plasmid within the bacterial cell [allows for the plasmid to be replicated in bacteria]

If bacteria containing non-recombinant pUC18 are plated on X-gal medium, would colonies appear blue or white or both?

blue

The ends left by some restriction enzymes that do not have 5' or 3' overhands are called what?

blunt ends

Efficient production of eukaryotic proteins in E. coli may require all of the following except? a) an E. coli expression vector with a Shine-Dalgarno sequence for efficient translation b) altered codon usage within the heterologous sequences to approximate the codon bias in E. coli c) a negative selectable marker to select against nonhomologous recombination events d) an E. coli expression vector with a promoter sequence that binds RNA polymerase e) the use of cDNAs, that are free of introns, as eukaryotic transgenes

c) a negative selectable marker to select against nonhomologous recombination events

Cloning animals by nuclear implantation involves all of the following except?

fertilization of the cell egg with a sperm cell followed by implantation in a surrogate mother

Which type of DNA library (genomic or cDNA) would include introns and promoters?

genomic cDNA does not have introns

In the pUC plasmids, a polylinker region that includes multiple restriction sites is embedded in the lacZ component. If a sequence of interest is inserted into this region, what is the effect on lacZ?

inactivation

What are the short single-stranded overhands created by cleavage of DNA by specific restriction enzymes?

sticky ends

Next, you would like study the gene that you ligated into the plasmid in Part B. From that experiment, which colonies should you select to culture for further experimentation?

white colonies that grew on complete media + ampicillin + X-gal Colonies that are white when grown on complete media + ampicillin + X-gal contain the plasmid with the gene ligated into the MCS and are the colonies that should be selected for culture for further experiments. The ampicillin in the media is used to select for cells that contain the plasmid which has an ampR gene. The X-gal in the media is used to select for cells that contain the plasmid and the gene of interest. If the gene of interest is present in the MCS, the lacZ gene will be disrupted and will not be able to produce a blue color in the presence of X-gal, and the colonies will be white.