restriction sites, to make it easier to insert foreign DNA. They also have other
ID: 265944 • Letter: R
Question
restriction sites, to make it easier to insert foreign DNA. They also have other useful characteristics, such as resistance to ampicillin, which makes it possible to select bacteria that have been transformed, and not the far greater number of bacteria that were not transformed in the same process. Here is a diagram of one of the earlier plasmids to be engineered: pBR322 Notice all the restriction enzyme cut sites, which allow foreign DNA to be easily inserted. HindIII EcoRI EcoRV Notice also the "amp" and "tet" sites. These allow the recipient bacteria to survive and reproduce even when grown on media containing the ampicillin or tetracyclin, respectively BamHI 4359 0 29 4000 375 Sal Pst 661 tet amp 1000 Usually, when we transform bacteria, i.e. get them to take in our plasmid, most of the bacteria will not take in the plasmid. Before we used plasmids with antibiotic resistance genes in them, it was necessary screen many individual colonies to find the few bacteria that were transformed. But if the plasmid has an antibiotic resistance gene on it, the pBR322 4361 bp ori 2000 2295 Ndel transformed bacteria are the only ones that will grow on medium with the antibiotic in it, simplifying the work enormously. 2 If you were to cut pBR322 with Hind III, what would happen? If you were to cut pBR322 with EcoRV at 185 bp (base pairs i.e. nucleotides) and Nde I at 2295 bp, how many fragments would you get, and how big would they be, in base pairs?Explanation / Answer
Ans A
If we cut the pBR322 with Hind III, then it will be linear DNA molecule, which can acceprt the forein molecule of DNA at the position of 29th no of base as shown in figure.
Now after insertion of forein gene, the plasmid will shows resistance against two antibiotic Amphicillin and tetracyclin. because insertion of foreign DNA doesn't distrubs any antibiotic gene. so both gene will express and both will show resistance, but similarly plasmid molecules without any foreign molecule insertion also shows resistanc for both the antibiotic. so it will create some problems at the selection time.
ANS B
By cutting pBR322 with EcoRV and Nde I,
there will be two fragments of Plasmid DNA,
Larger with 2251 bp and (Remaining fragment (total size 4361- smaller 2110bp))
smaller with 2110 bp. (2295 position of Nde I - 185 position of EcoR V)
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