In a molecular biology laboratory, a student obtained competent E. coli cells and used a common transformation procedure to induce the uptake of plasmid DNA with a gene for resistance to the antibiotic kanamycin. The results below were obtained. Plate I. LB agar +kan plasmid Plate II. LB agar with kanamycin +kan plasmid Plate III. LB agar no plasmid added Plate IV. LB agar with kanamycin no plasmid added During the course of an E. coli transformation laboratory, a student forgot to mark the culture tube that received the kanamycin-resistant plasmids. The student proceeds with the laboratory because he thinks that he will be able to determine from his results which culture tube contained cells that may have undergone transformation. Which plate would be most likely to indicate transformed cells? A plate with 100 colonies growing on LB agar without kanamycin A plate with 100 colonies growing on LB agar with kanamycin A plate with a lawn of cells growing on LB agar with kanamycin A plate with a lawn of cells growing on LB agar without kanamycin
Added by Willie O.
Close
Step 1
The student has introduced a plasmid with a gene for resistance to kanamycin into E. coli cells. This means that any E. coli cells that have successfully taken up this plasmid (i.e., undergone transformation) will be able to survive and grow in the presence of Show more…
Show all steps
Your feedback will help us improve your experience
Dominador Tan and 61 other Biology educators are ready to help you.
Ask a new question
Labs
Want to see this concept in action?
Explore this concept interactively to see how it behaves as you change inputs.
Key Concepts
Recommended Videos
In a transformation experiment, you mix E. coli competent cells with a small amount of pGFPuv plasmid DNA. After performing the heat shock and incubations according to the protocol, you then spread your cells on two separate agar growth media. One is a complete medium called "LB" that allows rapid growth of all cell types. The other is the same LB medium that has been augmented with the antibiotic ampicillin; it supports the growth of only those cells that have acquired an antibiotic resistance gene for ampicillin, presumably via a transformation procedure. A typical set of results appears within the support document that accompanies the transformation and PCR lab handout. Describe the bacterial growth that appears on the two plates shown in the support document. Which of the two plates, Plate 1 or Plate 2, contains the ampicillin antibiotic? Please explain your answer.
Qudsiya A.
GENE CLONING Questions and Experimental Observations 1. First examine and compare the growth of bacteria that received no DNA when plated to a plate containing no ampicillin to the plate that contained ampicillin. What is the phenotype of the original E. coli culture BEFORE transformation? In this experiment if they didn't take up a plasmid, were they ampicillin sensitive? 2. Count and record the number of colonies on the plates that were incubated with ’uncut’ DNA and ’cut’ DNA. If you have thousands of transformants, you can divide the plate into four or six sections. Count the number of transformants in one section and multiply by the number of sections to get a pretty good estimate. Type of DNA | Cut plasmid DNA | Uncut plasmid DNA Number of Colonies (White Light) | | Number of Colonies (UV Light) | | 3. The cut DNA represents linear DNA, while the uncut represents a circular plasmid. How does the transformation efficiency of the linear DNA compare to that of the circular plasmid? Can E. coli be transformed equally well with any form of DNA? 4. The growth on the ’No ampicillin’ No DNA plate gives an indication of the total number of cells plated. The colonies on the uncut DNA indicate the number of competent cells (those that took up the ampicillin resistance plasmid). Comparison of these two plates gives an indication of the percentage of the total bacteria that take up plasmid. If you didn't have ampicillin selection to help in identifying the bacteria that took up the plasmid, would they be easy to find? 5. What new phenotypes have been acquired by the transformed bacteria (those that take up the plasmid)? How have their appearance and ability to grow been changed?
Josee P.
DNA Cloning The plasmid cloning vector $\mathrm{pBR} 322$ (see Fig. $9-3$ ) is cleaved with the restriction endonuclease $P$ stl. An isolated DNA fragment from a cukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are sclected by growth in the presence of tetracycline. (a) In addition to the desired recombinant plasmid, what other types of plasmids might be found among the transformed bacteria that are tetracycline-resistant? How can the types be distinguished? (b) The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one cnd. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note that in $\mathrm{pBR} 322$, the Pst and EcoRI restriction sites are about $750 \mathrm{bp}$ apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted. FIGURE CANT COPY
Recommended Textbooks
Biology for AP Courses
Objective Biology for NEET
Introduction to General, Organic and Biochemistry
Transcript
18,000,000+
Students on Numerade
Trusted by students at 8,000+ universities
Watch the video solution with this free unlock.
EMAIL
PASSWORD