Question

Question 3(BOTANY)

You want to clone and express the gene that you’ve isolated from devil’s claw in E. coli. Describe all the necessary adjustments that you would need to make to your gene to ensure correct expression in E. coli.                                                                                                                                                                                 (5)

Answer 1

The gene needs to have all the elements to correctly replicate and be expressed in a bacterial cell, this is normally acquired when the gene is inserted into a proper vector. The vector needs an Ori site, which will work as the origin of replication, the vector also needs some restriction sites for the restriction enzymes to cut and insert the gene, and finally a selective marker is also good, so we can confirm the succesful cloning into the bacterial cells.

The isolated gene needs to be incubated with the vector, along with the correspondent restriction enzymes and ligases. The restriction enzyme will cut open the vector, while the ligases will attach the gene into the vector closing it up. The vector then is going to be inserted in the bacterial cells, electroporation will make the cells susceptible to recieve vectors, then the bacteria will be incubated in optimal conditions for their recovery. The final step is to confirm the success, we are going to grow the bacteria in the respective medium according to the selective marker, for example, if the marker is an antibiotic resistance gene this antibiotic will be used to grow the bacteria, succesfull formation of colonies confirms the successfull cloning. The bacteria will start to express the given gene