Genetics: From Genes to Genomes
6th Edition
ISBN: 9781259700903
Author: Leland Hartwell Dr., Michael L. Goldberg Professor Dr., Janice Fischer, Leroy Hood Dr.
Publisher: McGraw-Hill Education
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Chapter 14, Problem 24P
You can carry out matings between an Hfr and F strain by mixing the two cell types in a small patch on a plate and then replica plating to selective medium. This methodology was used to screen hundreds of different cells for a recombination-deficient recA- mutant. Why is this an assay for RecA function? Would you be screening for a recA mutation in the F or Hfr strain using this protocol? Explain.
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You can carry out matings between an Hfr and F strain by mixing the two cell types in a small patch on a plate and then replica plating to selective medium. This methodology was used to screen hundreds of different cells for a recombination-deficient recA - mutant. Why is this an assay for RecA function? Would you be screening for a recA mutation in the F or Hfr strain using this protocol? Explain.
After transformation you were asked to grow bacterial cells transformed with plasmid on a plate that had X-gal and ampicillin. X-Gal is often used as in indicator dye, which turns blue when metabolized by B-galactosidase protein and used to test if cloning experiments have worked. [Note look at the vector diagrams carefully]
Briefly explain how you would find the bacterial cells that are transformed with the plasmid with the YFG inserted.
Assume you have successfully cloned a small (200 bp) fragment of DNA into the polylinker region of a pUC18 cloning vector. Describe the appearance of transformed colonies you would expect to see on each of the following plates: plain media, media containing ampicillin, media containing tetracycline, media containing ampicillin and X-Gal.
Chapter 14 Solutions
Genetics: From Genes to Genomes
Ch. 14 - Choose the phrase from the right column that best...Ch. 14 - The unicellular, rod-shaped bacterium E. coli is 2...Ch. 14 - Now that the sequence of the entire E. coli K12...Ch. 14 - Bacterial genomes such as that of E. coli...Ch. 14 - List at least three features of eukaryotic genomes...Ch. 14 - Describe a mechanism by which a gene could move...Ch. 14 - High salt concentrations tend to cause protein...Ch. 14 - Recently, scientists tested the possibility that...Ch. 14 - A recent metagenomic study analyzed the...Ch. 14 - Linezolid is a new type of antibiotic that...
Ch. 14 - A liquid culture of E. coli at a concentration of...Ch. 14 - Pick out the medium i, ii, iii, or iv onto which...Ch. 14 - This problem concerns Fig. 14.14, which...Ch. 14 - In two isolates one is resistant to ampicillin,...Ch. 14 - E. coli cells usually have only one copy of the F...Ch. 14 - In E. coli, the genes purC and pyrB are located...Ch. 14 - DNA sequencing of the entire H. influenzae genome...Ch. 14 - Genes encoding toxins are often located on...Ch. 14 - a. You want to perform an interrupted-mating...Ch. 14 - In Problem 19, do you think that most of the...Ch. 14 - One issue with interrupted-mating experiments such...Ch. 14 - Prob. 22PCh. 14 - Starting with an F- strain that was prototrophic...Ch. 14 - You can carry out matings between an Hfr and F...Ch. 14 - Genome sequences show that some pathogenic...Ch. 14 - Generalized and specialized transduction both...Ch. 14 - This problem highlights some useful variations of...Ch. 14 - A researcher has a Trp auxotrophic strain of E....Ch. 14 - Streptococcus parasanguis is a bacterial species...Ch. 14 - The sequence at one end of one strand of the...Ch. 14 - Scientists who study amino acid biosynthesis...Ch. 14 - Suppose that you could obtain radioactively...Ch. 14 - Prob. 34PCh. 14 - Some scientists are trying to engineer...
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- Once cloning is complete, a recombinant OXA-M290 protein can be produced in E. coli. Competent E. coli BL21(DE3) cells are transformed with the pET-28a-OXA-M290 plasmid, and the transformed cells are grown on an agar plate that contains kanamycin. The E. coli BL21(DE3) cells transformed with the pET-28a-OXA-M290 vector are next grown in liquid growth media that contains kanamycin. Once the culture reaches an OD600 of 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5 mM to induce protein production. How does the addition of IPTG result in the transcription of genes that are regulated by the T7 promoter in E. coli BL21(DE3) cells? [4 – 6 sentences suggested] For Context ONLY: For this biology course and based on the results obtained from a previous BLAST search, the bacteria responsible for the infection at a Hospital is most likely a new strain of Escherichia coli. This strain is given the name E. coli AFB. Earlier, BLAST was used to study a…arrow_forwardblaA ori lacl Plac rrgB rrgA lacZYA Recombinant Plasmid "B"arrow_forwardBy conducting conjugation experiments between Hfr and recipientstrains, Wollman and Jacob mapped the order of many bacterialgenes. Throughout the course of their studies, they identified severaldifferent Hfr strains in which the F-factor DNA had been integratedat different places along the bacterial chromosome. A sample of theirexperimental results is shown in the following table:Analyze data. Compare and contrast. Make a drawing.arrow_forward
- X-gal is used to help with what we call “blue/white screening.” X-gal is a substrate that the enzyme beta-galactosidase can convert to blue color. When transformation reactions are plated on LB plates with (amp and) X-gal, if an insert was not cloned into the SmaI-site of pUC19, the bacteria will be blue. If an insert was cloned into the SmaI-site of pUC19, the bacteria will be white. Based on the information in this question, and shown in Figure 1 below, can you explain why we are hoping to obtain white colonies? (Please explain both why blue colonies lack a cloned insert, and why white colonies possess a cloned insert.) Fig. 1. Restriction map of pUC19 (2686 bp). Image obtained from the New England Biolabs e-catalog (https://www.neb.com/products/n3041-puc19 vector#Product%20Information_Properties%20&%20Usage). Note the presence of the lacZa gene, and note that the SmaI site (nt 412) into which (ideally) your insert was ligated is found within the lacZ ORF. Enzymes in the…arrow_forwardWhen using a conventional plasmid cloning vector containing a b-galactosidase gene, it is possible to perform a "blue-white screen" to determine which bacteria have taken up a plasmid into which a DNA fragment as been inserted, as opposed to those that have taken up just reclosed plasmid vector, by growing the transformed cells on nutrient agar plates containing the artificial b-gal substrate X-gal. Will bacteria that have taken up a plasmid into which a DNA fragment has been inserted form a blue colony or a white colony when grown on this medium? Briefly explain why these bacteria would form a colony of the color you chose.arrow_forwarda. Bacteriophage P22 was used in generalized transduction experiments to infect the Salmonella typhimurium donor strains described in the table below. The resulting phage lysates were then used to infect the S. typhimurium recipient strains listed in the table. In each cross, a phenotype was selected for one of the three genetic markers studied (str, aceA, thrA), and then replicates were performed to select the corresponding recombinants for the other two markers. The results are given in the following table: Recipient strain Selected phenotype Selected recombinants Donor strain str thrA aceAt thrA str aceA+ strs thrA+ aceA thrA+ str aceA Str Ace+ Str ThrA ThrA+ ThrA ThrA+ Ace Ace+ str: gene involved in streptomycin resistance, aceA: gene involved in the use of acetate as a carbon source, thrA: gene involved in the biosynthesis of threonine. Number 60 40 95 5 10 90 What are the selective media used in these three transduction experiments, on the one hand to obtain the selected…arrow_forward
- You have just carried out a transformation using a plasmid (possessing a Amp-resistance gene) that was 0.001 ug/ul in concentration. You added 10 ul of this plasmid to 100 ul of a bacterial cell suspension. Then you added 250 ul of LB following heat shock. After plating 200 ul of this cell suspension with LB onto a LBA plate, you observe 8 colonies on this plate. What is the transformation efficiency of this plasmid (in colonies/ug of plasmid) based on these results? about 1400 O about 80O about 4000 O about 400O O about 8000 f6 米 IO fs fg f1o 19 08. O OOarrow_forwardIn a generalized-transduction experiment, phages arecollected from an E. coli donor strain of genotype cys+leu+ thr+ and used to transduce a recipient of genotypecys- leu- thr-. Initially, the treated recipient populationis plated on a minimal medium supplemented with leucine and threonine. Many colonies are obtained.a. What are the possible genotypes of these colonies?b. These colonies are then replica plated onto threedifferent media: (1) minimal plus threonine only, (2)minimal plus leucine only, and (3) minimal. Whatgenotypes could, in theory, grow on these three media?c. Of the original colonies, 56 percent are observed togrow on medium 1, 5 percent on medium 2, and nocolonies on medium 3. What are the actual genotypes ofthe colonies on media 1, 2, and 3?d. Draw a map showing the order of the three genes andwhich of the two outer genes is closer to the middle genearrow_forwardPlease DESCRIBE, in outline form, the method you will use to select for bacterial cells that have taken up the pL311 plasmid, and to screen those cells for the presence of plasmids that are likely to contain a cloned gene. Be sure to mention the specific media you will use. In addition, please explain the rationale behind this specific selection and screening procedure. (Remember that you have available the following types of media: (i) media containing neither kanamycin nor X-gal, (ii) media containing BOTH kanamycin and X-gal, (iii) media containing tetracycline, and (iv) media containing ampicillin.arrow_forward
- A plaque assay is performed beginning with 1 mL of a solution containing bacteriophages. This solution is serially diluted three times by combining 0.1 mL of each sequential dilution with 9.9 mL of liquid medium. Then 0.1 mL of the final dilution is plated in the plaque assay and yields 17 plaques. What is the initial density of bacteriophages in the original 1 mL?arrow_forward7. Consider the following plasmid (size 6700 bp), with restriction sites at the positions indicated: BamHI + 1 6700 bp 2800 3500 EcoRI BamHI Probearrow_forwardIt is desired to isolate genomic DNA from liquid culture of S. cerevisiae yeast. A commercial kit will be used to isolate genomic DNA from this liquid culture. Answer the following questions to understand the strategy used by commercial kits for genomic DNA isolation. a) List all the steps from cell pellet preparation to DNA elution. b) With which feature can the membrane in the column that comes with the commercial kit bind DNA? c) Which component in the kit would you use to recover the DNA from the membrane of the column to which the DNA was attached?arrow_forward
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