Practice Questions

TLS Online TPP Program

#Question id: 13091

To express a yeast gene in E. coli, your task is to design a strategy to insert the yeast gene into the bacterial plasmid. Below is a map of the area of the yeast genome surrounding the gene in which you are interested.

The distance between each tick mark placed on the line above is 100 bases in length
Below are the enzymes you can use, with their specific cut sites shown 5’-XXXXXX-3’ 3’-XXXXXX-5’

The plasmid is 5,000 bases long and the two farthest restriction enzyme sites are 200 bases apart. The plasmid has an ampicillin resistance gene somewhere on the plasmid distal from the restriction cut sites.

You do the digestion of the insert and the vector and then ligate the two digestions together. You then transform the ligation into bacteria and select for ampicillin resistance. You get three colonies on your transformation plate. You isolate plasmid from each one and cut each plasmid with the enzyme XbaI. You then run your three digestions on an agarose gel and see the following patterns of bands. Describe what each plasmid actually was that was contained in each of the three colonies.

Which colony’s plasmid do you actually want to use for your studies?

#SCPH06 I Botany

colony-1 because you want the bacterial promoter to drive transcription of the yeast gene in the correct orientation
colony-2 because you want the bacterial promoter to drive transcription of the yeast gene in the correct orientation
colony-3 because you want the bacterial promoter to drive transcription of the yeast gene in the correct orientation
All colonies because the correct strand of DNA is used as a template in transcription.