1. We can construct our gene library by cloning the PstI fragments into either p
ID: 79211 • Letter: 1
Question
1. We can construct our gene library by cloning the PstI fragments into either plasmid pBR322 or phage lambda. Which one should we choose and why?
2. Suppose we want to insert the PstI fragments into the Bam HI site within the gene for tetracycline resistance (tetr) in plasmid pBR322. If both of these restriction enzymes produce cohesive ends, how can this be done?
3. After transforming E.coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing DNA insert)?
4. If a million tetracycline-sensitive, ampicillin-resistance clones are grown on nutrients agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?
5. After selecting a clone carrying the gene for protein P, its ells are propagated to high density in nutrient broth. The chimeric plasmid (with an insert of the gene for protein P) is then extracted and purified from the rest of the cellular DNA. How can we now isolate the gene from the plasmid?
6. How can we demonstrate that the gene we have isolate is indeed the one for protein P?
Explanation / Answer
1.pBR322 is a commonly used plasmid cloning vector in E. coli (4.361 kbp in length) while Phage Lambda is a double stranded virus that can infect E.coli. Plasmid vectors can carry insert of size upto 15 kbp while lambda phage can carry upto 25 kbp. Since the insert size is usually small, we can procede with pBR322.
2. To be able to clone PstI fragments into the Bam HI site within the gene for tetracycline resistance (tetr) in plasmid pBR322, both have to be treated with two restriction enzymes that create compatible ends sequentially before insertion.
3.The E.coli with chimeric plasmids express gene for tetracycline resistance. If you do replica plating in media with and without tetracycline, we can indentify the colonies that are tetracycline resistant therefor indentify the plasmid.
4.To detect rare clones that contain gene P, we plate it on tetracycline containing media plates then select for the protein P.
5. We perform a restriction digestion on the plasmid at the site of insertion of the gene P and run it on an agarose gel and isolate the gene.
6. After isolation, we transform it again on another expression plasmid and isolate the protein of interest and do mass spectometry to indentify the protein.
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