3. Why did we need the reporter gene in this experiment? That is what would have happened if there were no reporter gene on the plasmid (1 pt)? 4. Explain the importance of the antibiotic ampicillin in the growth medium (1 pt). What would happen if the medium didn't contain ampicillin (1 pt)? 5. What are competent cells (1 pt)? Additionally, explain what heat-shocking and cold-shocking did to the cells (2 pts). Then explain the role of $CaCl_2$ in this experiment (1 pt). 6. Explain the term \"satellite colonies\" (1 pt).
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It allows us to visually observe the expression of the gene of interest by producing a detectable signal, such as fluorescence or color change. Show more…
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Ampicillin (AMP) resistance is a selectable marker located on the pGLO plasmid. What is the purpose of the selectable marker? What is the "bla" gene, and how does it relate to ampicillin resistance? What component of the LB/AMP/ARA media is selective? What does it select for? How is this visualized on the media? The reporter gene on the pGLO plasmid is GFP. What does "GFP" stand for? What is the purpose of the reporter gene? What does it tell you about the organism? How does arabinose regulate the expression of GFP? What would you EXPECT to see on the +pGLO LB/AMP plates if there was NO arabinose? What are some possible explanations to why you may see colonies growing on the +pGLO LB/AMP/ARA plates that are NOT glowing? The -pGLO tube was plated on the LB/AMP/ARA media. Why was there no growth on these plates? What would growth indicate on these plates? On the LB/AMP/ARA plates: What indicates successful transformation? (How is this visualized?) What indicates successful gene expression? (How is this visualized?)
Sri K.
b) A gene for a rhodopsin protein ("RHO") was ligated into an E. coli bacterial plasmid that already contain the gene for ampicillin resistance (ampᄒ). This plasmid was mixed with an E. coli bacterial sample; as a control, wild type E. coli bacteria (without the RHO/ampᄒ plasmid) was also maintained. Both samples were plated on nutrient agar, half of which were supplemented with the antibiotic, ampicillin, and the other half were not. The results of E. coli growth are shown. Dots indicate growth of bacteria. Answer these questions based on this experiment: i) Which plate(s) have some ampicillin-resistant bacteria growing: I / II / III / IV ? ii) Which plate(s) contain some recombinant RHO gene plasmids: I / II / III / IV ? iii) Which plate(s) contain only bacteria with recombinant plasmids: I / II / III / IV ? iv) What was the advantage of using a plasmid with the ampᄒ gene as a vector for cloning the RHO gene? v) What is the purpose of the wild-type bacterial plates (I & II) in this experiment? vi) What is the purpose of growing bacteria on non-ampicillin plates (I & III) in this experiment?
Supreeta N.
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.
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