A student designs four cloning vectors. Each has a fault…
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A student designs four cloning vectors. Each has a fault. Name the missing or wrong feature of each, and say what would go wrong in the laboratory.
Vector 1: it has an origin of replication and three sites for the enzyme EcoRI. It has an ampicillin resistance gene.
Vector 2: it has an ampicillin resistance gene and a single EcoRI site. It has no origin of replication.
Vector 3: it has an origin of replication and a single EcoRI site. It has no selectable marker.
Vector 4: it has an origin of replication, a kanamycin resistance gene and a single EcoRI site. The student plates the host cells on a medium with ampicillin.
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Work it out on paper first. It is fine to get stuck or make a mistake: that is how we learn. When you are ready, go through the solution one step at a time, and check each step against your own.
Given. Four vectors.
Plan. A vector needs an origin of replication, a selectable marker and very few cloning sites. Check the features one by one.
Step 1. Vector 1. It has three EcoRI sites. EcoRI would cut the vector into three pieces, and cloning would become hard. A good vector has one site.
Step 2. Vector 2. It has no origin of replication. The vector cannot copy itself in the host. It is lost as the cells divide, or it makes no copies of the gene.
Step 3. Vector 3. It has no selectable marker. After the host cells are treated, there is no way to tell which cells took up the vector.
Step 4. Vector 4. The marker is a kanamycin resistance gene, but the medium holds ampicillin. The cells that took the vector have no protection against ampicillin, and they die with the others.
Answer: Vector 1: too many sites. Vector 2: no ori. Vector 3: no marker. Vector 4: marker and antibiotic do not match.
Check. Each vector breaks one of the three requirements, or uses the marker wrongly.
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