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A plasmid that contains genes that provide ampicillin resistanceand tetracycline resistance is cut with EcoRI, which cuts insidethe ampicillin resistance gene. The cut plasmid is combined withEcoRI digested genomic DNA from Drosophila in the presence ofligase and the mixture is finally used to transform E. coli.

a. Which antibiotic should be used to select for cells that haveincorporated the plasmid?

b. What will be the antibiotic resistance/sensitivity genotypefor cells that have incorporated the plasmid with no insert?

c. What will be the antibiotic resistance/sensitivity genotypefor cells that have incorporated the plasmid containing aninsert?

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Jean Keeling
Jean KeelingLv2
28 Sep 2019
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Related questions

An ampicillin-resistant, tetracycline-resistant plasmid iscleaved with EcoRI, which cuts within the tetracycline gene. Thecut plasmid is ligated with EcoRI-digested Drosophila DNA toprepare a genomic library. This DNA is then used to transform E.coli K12. A) Under what conditions should you grow bacteria inorder to select for transformed cells (those that have taken up aplasmid)? B) How can you identify bacteria carrying a plasmid thatcontains a fragment of the Drosophila DNA? C) How can you explainthe presence of colonies that are resistant to bothantibiotics?

A plasmid that carries two antibiotic resistance genes, to ampicillin and tetracycline, respectively, has one restriction site for EcoRI located within the tetracycline resistance gene. Eukaryotic DNA containing a gene of interest (g.o.i.) has been digested with diffferent restriction enzymes, none of which cut within the gene of interest sequence). The resultant fragments are added to plasmid DNA that has been digested with EcoR1 only; the mixture was then incubated with DNA ligase. Resulting plasmids (recombinant and otherwise) are added to competent bacteria lacking resistance genes and growing in nutrient broth. The bacteria are then plated and allowed to grow overnight producing isolated colonies. Samples of these colonies are then grown in four different media: nutrient broth plus ampicillin; nutrient broth plus tetracycline; nutrient broth plus ampicillin and tetracycline; and nutrient broth containing no antibiotics.

1. The bacteria containing the plasmid with the gene of interest successfully insertedin would grow in

A. Tthe nutrient broth only.

B. The nutrient broth, the ampicillin broth, and the tetracycline broth.

C. Tthe nutrient broth and the tetracycline broth only.

D. The ampicillin broth and the nutrient broth.E.Tthe ampicillin and tetracycline broth only.

2. Bacteria transformed with the plasmid alone (not containing any eukaryotic DNA), would grow

A. Only in the broth containing both antibiotics.

B. In the nutrient broth plus ampicillin, but not in the broth containing tetracycline.

C. In the broth containing tetracycline, but not in the broth containing ampicillin.

D. Only in the broth that contained no antibiotics.

E. In all four types of broth.

4. Bacteria that did not take up any plasmids at all would grow in which media?

A. The tetracycline and ampicillin broth only

B. The nutrient broth and the tetracycline broth only

C. The nutrient broth and the ampicillin broth only

D. The nutrient broth only

E. All four broths

1. Below is a map of a plasmid called pBR322:

(Cannot figure out how to post the picture)

Suppose that the pBR322 plasmid is cleaved with the restriction enzyme PstI. Suppose a PstI-digested segment of mouse DNA is then ligated into the PstI site in pBR322. This recombinant DNA molecule is then used to transform E. coli cells. a. Which antibiotic should be added to the medium to select for cells that have incorporated a recombinant plasmid? b. How can you explain the presence of bacterial colonies that are resistant to both antibiotics? c. You decide to digest your recombinant DNA molecule with BamHI and EcoRI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel? d. You decide to digest your recombinant DNA molecule with BamHI and PstI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel? ?a. Which antibiotic should be added to the medium to select for cells that have incorporated a recombinant plasmid?

b. How can you explain the presence of bacterial colonies that are resistant to both antibiotics?

c. You decide to digest your recombinant DNA molecule with BamHI and EcoRI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel?

d. You decide to digest your recombinant DNA molecule with BamHI and PstI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel?

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