pBR322 is one of the first known cloning vectors to be used for molecular cloning. Similarly to pUC18, it has an ampicillin gene but in contrast, it has another gene known as tetracycline. These two genes confer resistance to their respective antibiotic, but suppose a foreign DNA fragment was introduced into the tetracycline gene. Would the tetracycline gene still be functional? How could you determine recombinant clones from non-recombinant clones?
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A plasmid (pBR322) that encodes resistance to ampicillin and tetracycline is digested with the restriction enzyme BamHI, which cuts the plasmid at a single site in the tetracycline resistance gene. The DNA is then annealed with BamHI digest of human DNA, ligated, and used to transform E. coli cells. You would put ampicillin in the agar plate to ensure that the cells of the bacterial colony contain the plasmid. How can you select the clones that contain the human DNA? Explain what you would do and what the results would be if the clone contained human DNA.
Tracy L.
You want to clone gene A (1000 bp) into the SalI restriction site of the vector pBR322 illustrated below. pBR322 contains both ampicillin and tetracycline resistance genes. a) Which antibiotics would you include in the agar plates that would allow bacterial cells to grow that have been transformed with: i) No plasmid. ii) Non-recombinant pBR322. iii) Recombinant pBR322 with gene A cloned into the SalI site. b) Assume the cloning of gene A was successful, and you have isolated the recombinant plasmid. You set up separate restriction enzyme digestion for the non-recombinant and recombinant plasmids with: (i) SalI (ii) PstI + NdeI. Draw the gel profile you expect to observe when you subject the products of these four reactions to agarose gel electrophoresis. Indicate the sizes of all bands on the gel.
Suman K.
d) A plasmid used as a cloning vector has the reporter genes tetR (tetracycline resistance) and lacZ (̢-galactosidase) with restriction sites for cloning DNA fragments within lacZ. What should be added to the growth medium to select for bacterial cells with recombinant vectors, and what color will the colonies of recombinant cells be? a. Tetracycline and X-Gal; blue b. Tetracycline and ̢-galactosidase; white c. Tetracycline and X-Gal; white d. X-Gal only; blue e. Tetracycline only; white e) Suppose that you want to express a protein from animal cells using recombinant DNA technology. Would you use a bacterium, virus, or yeast? Explain your answer. f) Cloning of a human growth hormone required construction of a cDNA in order to be clone it into a bacterial host cell. What is a cDNA and why was this strategy possibly used? Explain.
Adi S.
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