Biology: The Dynamic Science (MindTap Course List)
4th Edition
ISBN: 9781305389892
Author: Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher: Cengage Learning
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Chapter 17, Problem 14TYK
Summary Introduction
To review:
The experiment that can confirm the presence of a wild type bio+ allele in the plasmid of chromosomal DNA (deoxyribonucleic acid).
Introduction:
Minimal media is the media that only provides minimum requirement that is only inorganic salts and no amino acids. The colonies whose genome is complete, that is, they have genes for all requirements grows on the minimal media while others do not.
Electroporation is the technique in which electric pulse is given to the culture leading the opening of the transient pores in the cells that can allow the entry of the DNA from the surrounding.
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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.
you have transformed E.coli cells with a plasmid containing the gene for the enzyme beta-lactamase you have used 3µl of a stock solution of 2.5ng/µl DNA. You transformed 150/µl of competent cells, using the calcium chloride method. you have added 600µl of luria broth to the cells and then plated out 200µl on triplicate plates. upon counting the colonies, you got the following results:
plate 1: 120 cfu
plate 2: 35 cfu
plate 3: 95 cfu
please answer the following question:
1. what new characteristic will the cells have ater transformation?
2. how are you going to test for the new characteristic?
3. calculate the transformation efficiency of the experiment?
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
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Chapter 17 Solutions
Biology: The Dynamic Science (MindTap Course List)
Ch. 17.1 - What are the properties of F+, F-, and Hfr cells...Ch. 17.1 - Prob. 2SBCh. 17.2 - Prob. 1SBCh. 17.2 - How does viral infection of an animal cell and a...Ch. 17.2 - Prob. 3SBCh. 17.3 - Prob. 1SBCh. 17 - Prob. 1TYKCh. 17 - Prob. 2TYKCh. 17 - Which of the following is not correct for...Ch. 17 - Prob. 4TYK
Ch. 17 - Prob. 5TYKCh. 17 - Prob. 6TYKCh. 17 - When a virus enters the lysogenic stage: a. the...Ch. 17 - An infectious material is isolated from a nerve...Ch. 17 - Prob. 9TYKCh. 17 - Prob. 10TYKCh. 17 - Prob. 11TYKCh. 17 - Discuss Concepts As a control for their...Ch. 17 - Prob. 13TYKCh. 17 - Prob. 14TYKCh. 17 - Prob. 15TYKCh. 17 - Prob. 1ITD
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- The Molecular Cell Biology Unit of University X is working on a particular gene segment called ABC20. For a specific kind of experiment, they need to quantify the gene and require a lot of copies of the gene. The initial gel run showed a very faint band of the gene which made the quantification process difficult to proceed. Now propose a method with the help of which they can generate many copies of the gene. Here you need to know that they have the plasmid from where they can get the gene. You do not need to explain the process in detail rather answer in no more than 10 sentences on what can be done about this problem!arrow_forwardYou are about to isolate a 3000 bp large plasmid from an E.coli culture. You know that the plasmid is present in 100 copies per E. coli cell. You aim to have a final plasmid concentration of 100 ng/µl in a total volume of 50 µl. Assuming the yield is 100 %, how many E. coli cells should the culture from which the plasmid is to be isolated at least containarrow_forwardWhen plasmids are isolated from bacterial cells, they may existin a number of forms.a. List the different forms that may be found.b. Which do you think would migrate the fastest and farthest in anelectrophoresis experiment and why?arrow_forward
- You transform bacteria with a plasmid carrying the ampicillin-resistance gene ampR. How would you determine which bacteria took up the plasmid? O Bacteria containing the plasmid would be able to grow in the absence of ampicillin. O Bacteria containing the plasmid would be able to grow in the presence of ampicillin. O Bacteria containing the plasmid would be able to produce ampicillin. O Ampicillin-resistance is necessary for the plasmid DNA get into the bacteria.arrow_forwardblaA ori lacl Plac rrgB rrgA lacZYA Recombinant Plasmid "B"arrow_forwardYou have two cell cultures, each containing a different plasmid. The first plasmid is ~5kbp long and contains three 5'-TCGA-3' and the other is the same size, but only contains two 5'-TCGA-3' sequences. You forget to label the two cell cultures you grew and have to figure out which one contains each plasmid. How would you go about identifying the the two cultures.arrow_forward
- You are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), HindlII (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. H. E+H E+X H+X Kb +4.3 -2.8 -2.5 - - -2.0 -+1.8 - +1.5 +1.0 F0.8 12 a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forwardYou are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), HindlII (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. E H E+H E+X H+X Kb +4.3 +2.8 +2.5 - +2.0 -- -1.8 -1.5 12 +1.0 +0.8 +0.5 - a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forwardYou are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), Hindlll (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. +18 a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forward
- You have two E. coli strains, XL10 and K12. XL10 is ampicillin resistant, or AmpR, and can grow on lab media plates in the presence of ampicillin. K12 is ampicillin sensitive, or AmpS, and cannot grow on lab media plates in the presence of ampicillin. You suspect that the AmpR gene is carried on a plasmid in XL10, rather than on its chromosome, and want to design an experiment to test this hypothesis. In designing your experiment, you may assume that you have access to the following materials: all reagents, plates, plasmids, and equipment used in the lab this semester Overnight cultures of XL10 & K12 Competent cells of XL10 & K12 a. Describe briefly the steps of a simple experiment that you could perform to test the hypothesis that the AmpR gene is carried on a plasmid in XL10 and not on its chromosome. (NOTE: no need for detailed procedure, simply state what you would do is sufficient). b. What specific experimental result will tell you that your hypothesis is correct?arrow_forwardScientists modified the tumor-inducing (Ti) plasmid, a naturally occurring plant plasmid found in Agrobacterium tumefaciens, to create a tool used to introduce any gene of interest into plant cells. They created a binary system because a single Ti plasmid is too large to be easily manipulated. One part of the system is a disarmed plasmid, and the second part is a transformation vector. The following sentences describe the function of key DNA elements in the system. Virulence region Genes for conjugative transfer Disarmed Ti plasmid (T-region removed) ori Kan selectable marker plant selectable marker constitutive promoter T-region conjugative transfer virulence Kan (bacterial selectable marker) Amp selectable marker Plant selectable marker (e.g., herbicide resistance) Constitutive promoter T-DNA left border Place the terms in the appropriate blanks to complete the sentences. Not all terms will be used. 3' transcriptional terminator MCS (inserted gene of interest) T-region Transformation…arrow_forwardA plasmid vector has two genes in it, a gene for streptomycin resistance (strR) and another for kanamycin resistance (kanR). There is a restriction site within the strR gene that you are hoping to move your gene of interest into. After cutting both your vector and insert you mix them together in a test tube. You then transform your mixture into E. coli and plate your cells on a master plate without any antibiotics. After colonies appear, you pick and re-plate onto a streptomycin containing plate and onto a kanamycin containing plate. 1. Using those same pieces of short DNA that you created above, show me what one of them may look like if it was cut with a blunt end restriction endonuclease. With the other example, show me what it would look like if it was cut with a sticky end restriction endonuclease. 2. Why do restriction endonucleases exist in bacterial cells? What must they do to protect their own DNA?arrow_forward
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