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 9, Problem 19P
The lacZ gene from E. coli encodes the enzyme β-galactosidase, which can catalyze the conversion of a colorless compound called X-gal into a blue product. Molecular biologists have taken advantage of this property by constructing plasmid vectors that contain the lacZ gene with an EcoRI site in its middle (see figure that follows). After cutting this vector with the EcoRI enzyme, scientists ligate it together with EcoRI-digested human genomic DNA, transform the resultant molecules into ampicillin-sensitive E. coli cells, and plate these cells on petri plates containing ampicillin and X-gal. Some of the colonies growing on this plate are white in color, while others are blue. Why?
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Shown below is a diagram for a plasmid vector you want to use to clone a gene. The diagram shows the location of
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E B
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The human hexokinase enzyme has the same function as the bacterial hexokinase enzyme but is somewhat different in its amino acid sequence. You have obtained a mutant bacterial strain in which the gene for hexokinase is missing. If you introduce into your mutant strain a DNA plasmid engineered to contain the DNA coding sequence of the human hexokinase gene, what must you also include?
a)The human hexokinase promoter
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Explain the basis for the following statement: Transcription of two genes on a plasmid can occur without the concomitant action of a topoisomerase, but only if those two genes are oriented in opposite directions.
Chapter 9 Solutions
Genetics: From Genes to Genomes
Ch. 9 - Match each of the terms in the left column to the...Ch. 9 - For each of the restriction enzymes listed below:...Ch. 9 - The calculations of the average restriction...Ch. 9 - The DNA molecule whose entire sequence follows is...Ch. 9 - Why do longer DNA molecules move more slowly than...Ch. 9 - Agarose gels with different average pore sizes are...Ch. 9 - The following picture shows the ethidium...Ch. 9 - The linear bacteriophage genomic DNA has at each...Ch. 9 - Consider a partial restriction digestion, in which...Ch. 9 - The text stated that molecular biologists have...
Ch. 9 - a. What is the purpose of molecular cloning? b....Ch. 9 - a. DNA polymerase b. RNA polymerase c. A...Ch. 9 - Is it possible that two different restriction...Ch. 9 - A plasmid vector pBS281 is cleaved by the enzyme...Ch. 9 - A recombinant DNA molecule is constructed using a...Ch. 9 - Suppose you are using a plasmid cloning vector...Ch. 9 - Prob. 17PCh. 9 - The lacZ gene from E. coli encodes the enzyme...Ch. 9 - Your undergraduate research advisor has assigned...Ch. 9 - Which of the enzymes from the following list would...Ch. 9 - You use the primer 5 GCCTCGAATCGGGTACC 3 to...Ch. 9 - a. To make a genomic library useful for sequencing...Ch. 9 - Problem 15 showed part of the sequence of the...Ch. 9 - Eukaryotic genomes are replete with repetitive...
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- A plasmid, pUC18, contains the ampicillin-resistance gene, the origin of replication, and the ß - gal gene, which codes for the B-galactosidase protein. This protein can break down the synthetic chemical X-gal, producing a blue product that stains the entire cell blue (but is harmless to the bacteria). At the beginning of the B-gal gene there are several unique restriction sites (some of them are shown in the diagram below). You wish to clone a 1.0-kb Xbal fragment into the pUC18 plasmid, so you cut the plasmid with Xbal and, after removing the enzyme, mix the Xbal-cut plasmid with the 1.0-kb fragment, ligate, and transform competent bacteria. Pati Xbal EcoRI B-gal A Amp ori Figure: pUC18 plasmid map (a) On what medium would you grow your transformed bacteria? (b) Do you expect the bacteria carrying plasmid pUC18 (without the insert) to be blue or white when grown in the presence of X-gal? Explain.arrow_forwardYou receive purified mRNA that codes for the spike protein of SARS-CoV-2 as well as plasmid pET32(a+). Explain how you will prepare SARS-CoV-2 spike cDNA and name the enzymes that you will need how you will clone the cDNA into pET32(a+) and select the recombinant plasmid how you will then produce a recombinant SARS-CoV-2 spike and purify itarrow_forwardTransposon mutagenesis was used to generate a library of mutants within the Salmonella genome. You are trying to identify a colony with the transposon inserted in the pathogenic related gene SPI-1 using PCR. Forward and reverse primers are generated that flank either side of the gene and yield a wild type product that is 900 bases in length. Which of the colonies sampled in the gel would you expect to contain the SPI-1 gene with transposon insertion? 3,000 2,000 1,000 700 500 300 100 Ladder Colony A Colony B Colony C Colony D Colony E none colonies A&C colonies B&E O colonies A, C, &D colonies B, D, &E -arrow_forward
- A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. One of the most common types of shuttle vectors is the yeast shuttle vector. Examples of such vectors derived from yeast are Yeast Episomal Plasmid (YEp), Yeast Integrating Plasmid (YIp) and Yeast Replicating Plasmid (YRp). Among these three vectors, YIp has the lowest transformation frequency and copy number per cell. Explain why Ylp is still popularly used despite its limitations.arrow_forwardE. coli strains diploid for the lac region were constructed by introducing a plasmid carrying the lac genes. The plasmid carries one copy of the lac region, and the chromosome carries the other copy. The two copies of the lac region have different genotypes, as shown in the chart below. Indicate whether the products of the lacy gene (permease) and the lacZ gene (B-galactosidase) will be inducible, uninducible, or constitutive in each strain (assuming glucose is absent). lac region on plasmid lac region on chromosome permease B-galactosidase I-o+Z+Y- I+o+ Z-Y+ I+o+Z+Y- I+o° Z=Y+ I- oº Z+Y- I+o+ Z-Y+ Is o+ Z+Y- I+o+ Z-Y+ I+ oc Z+Y- IS O+ Z-Y+arrow_forwardIn E. coli, the gene bioD+ encodes an enzyme involved in biotin synthesis, and galK+ encodes an enzyme involved in galactose utilization. An E. coli strain that contained wild-type versions of both genes was infected with P1 phage, and then a P1 lysate was obtained. This lysate was used totransduce (infect) a strain that was bioD− and galK−. The cellswere plated on a medium containing galactose as the sole carbonsource for growth to select for transduction of the galK+ gene.This medium also was supplemented with biotin. The resultingcolonies were then restreaked on a medium that lacked biotin tosee if the bioD+ gene had been cotransduced. The following resultswere obtained:What topic in genetics does this question address?arrow_forward
- In E. coli, the gene bioD+ encodes an enzyme involved in biotin synthesis, and galK+ encodes an enzyme involved in galactose utilization. An E. coli strain that contained wild-type versions of both genes was infected with P1 phage, and then a P1 lysate was obtained. This lysate was used totransduce (infect) a strain that was bioD− and galK−. The cellswere plated on a medium containing galactose as the sole carbonsource for growth to select for transduction of the galK+ gene.This medium also was supplemented with biotin. The resultingcolonies were then restreaked on a medium that lacked biotin tosee if the bioD+ gene had been cotransduced. The following resultswere obtained:What information do you know based onthe question and your understanding of the topic?arrow_forwardThe following table lists 4 bacterial strains that are partial diploids for lac operon genes. Given the activity of beta-galactosidase measured for each strain in the absence (-lac) or presence (+lac) of lactose, complete the table by choosing the appropriate symbol (+, -, C, S) to indicate the allele of the gene or site missing from the table (blue numbers). strain A BC 5 C D 7 chromosome I O 1 2 4 1 [Select] 9 3 [Select] [Select] [Select] 9 [Select] + + Z + + 6 + I +5 + 10 plasmid O 3 + 7 C Z + 8 8 2 [Select] 4 [Select] 6 [Select] B-gal act. -lac +lac 0.002 0.003 0.002 0.058 0.063 0.121 0.059 0.062 Select] 1 ✔ [ Select] + is C Sarrow_forwardConsider three genes in E. coli: thr+, ara+, and leu+ (which give the cell the ability to synthesize threonine, arabinose, and leucine, respectively). All three of these genes are close together on the E. coli chromosome. Phages are grown in a thr+ ara+ leu+ strain of bacteria (the donor strain). The phage lysate is collected and used to infect a strain of bacteria that is thr− ara− leu −. The recipient bacteria are then tested on selective medium lacking leucine. Bacteria that grow and form colonies on this medium (leu+ transductants) are then replica-plated on medium lacking threonine and on medium lacking arabinose to see which are thr+ and which are ara+. Another group of the recipient bacteria are tested on medium lackingthreonine. Bacteria that grow and form colonies on this medium (thr+ transductants) are then replica-plated on medium lacking leucine and onto medium lacking arabinose to see which are ara+ and which are leu+. Results from these experiments are as follows:…arrow_forward
- Now that you’ve isolated the gene and made lots of copies, you need to insert the gene into something you can manipulate and move into your lab strain of E. coli. You have access to the following vectors, either pBR322 or pUC19 (look them up here to see a map https://www.neb.com/products/dna-plasmids-and-substrates Please note the restriction sites in BOLD appear only once in the plasmid). You may use either vector. What is a vector? Which vector did you choose? Explain why you made that choice. What enzyme(s) will you use to place your fragment into the vector? Explain why you chose these enzymes. How will you move this construct into coli? What phenotype will tell you if the coli have taken up the plasmid? What phenotype will tell you if the coli have taken up the plasmid with the gasP gene?arrow_forwardF ′strains in E. coli are derived from Hfr strains. In some cases, these F ′strains show a high rate of integration back into the bacterial chromosome of a second strain. Furthermore, the site of integration is often the site occupied by the sex factor in the original Hfr strain (before production of the F ′strains). Explain these results.arrow_forwardA plasmid that is both ampicillin and tetracyclineresistant is cleaved with PstI, which cleaves within theampicillin resistance gene. The cut plasmid is ligated withPstI-digested Drosophila DNA to prepare a genomic library,and the mixture is used to transform E. coli K12. Question: How can you explain the presence of colonies thatare resistant to both antibiotics?arrow_forward
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