Blue white screening with plasmid pUC19: 1. The plasmid and foreign DNA containing the gene of interest are cut with the same __________________ enzyme. Why? 2. The foreign DNA will insert into the __________________ site, disrupting the lacZ gene encoding ?- galactosidase. The bacteria that receives this __________________ (hint: new hybrid) plasmid can/cannot (circle one) hydrolyze X-gal (chromogenic substrate for ?-galactosidase). Thus, these bacterial colonies appear __________________ in color. a. In nature, what substrate is cleaved by ?-galactosidase? 3. The vector contains a gene that confers resistance to the drug __________________. a. When grown under selective conditions (i.e. media supplemented with this antibiotic), bacteria that contain the plasmid are resistant/sensitive (circle one) to this antibiotic. 4. Bacteria that are __________________ in color do not contain plasmid inserted with foreign DNA. Why do they still grow under selective conditions?
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A geneticist uses a plasmid for cloning that has the lacZ gene and a gene that confers resistance to penicillin. The geneticist inserts a piece of foreign DNA into a restriction site that is located within the lacZ gene and uses the plasmid to transform bacteria. Explain how the geneticist can identify bacteria that contain a copy of a plasmid with the foreign DNA. Bacteria with the correct plasmid will produce the only living colonies on selective media containing penicillin and X-gal. Bacteria with the correct plasmid will produce white colonies on selective media containing penicillin and X-gal. Bacteria with the correct plasmid will produce blue colonies on selective media containing penicillin and X-gal. Bacteria with a plasmid containing foreign DNA cannot be identified until the bacterial cells are lysed and the DNA extracted and analyzed. Bacteria with the correct plasmid will produce both blue and white colonies on selective media containing penicillin and X-gal.
Jenny W.
Suppose we have a plasmid as shown below that contains an ampicillin resistant gene (amp) and a gene that produces a blue color (lacZ). Bacteria will appear blue if they contain an intact lacZ gene and are grown in the presence of a compound X-gal. If the lacZ gene is disrupted, the colonies will remain white. Describe how we would use this to insert our gene of interest and test for the presence of the gene. We will be inserting the gene into the lacZ gene, so describe which restriction enzyme we would use and what should be added to the bacterial growth media to test for the plasmid uptake as well as the presence of our gene of interest.
Shaiju T.
Figure 1 (see below) shows the plasmid pUC18. This plasmid contains a LacZ gene interrupted by a multiple cloning site (MCS), a gene conferring resistance to ampicillin (AmpR) and an origin of replication (Ori). MCS Lac Z gene AmpR Ori Figure 1: Plasmid pUC 18 map. 1) Colonies derived from bacteria transformed by this plasmid accumulate a blue product in the presence of Xgal. Xgal is a colorless substrate of Ě -galactosidase (an enzyme coded by the LacZ gene) into a blue product. Untransformed bacteria would form white colonies in the presence of Xgal. a) Does the insertion of the multiple cloning site affect the production of active Ě -galactosidase? Explain 3 pts b) What is the likely color of colonies transformed by a pUC18 plasmid that contain a piece of DNA cloned at the MCS? Explain 2 pts c) What is the role of the AmpR gene in the plasmid? 4 pts d) What is the role of the origin of replication in the plasmid? 4 pts
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