Question

Questions 1

You are the principle investigator of a research unit in the Botany and Plant Biotechnology department that studies medicinal plant usage in South Africa. Your work focused extensively on devil’s claw. You have identified that one of the plant extracts, harpagoside that is an iridoid glycoside, is of medicinal use. You would like to produce this iridoid glycoside in a liquid culture of Escherichia coli at commercial scale. You have identified that the key enzyme that you would need to introduce into E. coli is an iridoid synthase from devil’s claw. This gene is known and studied in Arabidopsis thaliana but remains to be identified from devil’s claw. How would you go about using the known Arabidopsis sequence to isolate the full length gene from devil’s claw? Describe the steps that you will follow in detail and provide all the details of the techniques that you use in each step and how your approach will impact on these steps. Hint: It is critical to remember that devil’s claw is a eukaryote and E. coli, where you want to express the iridoid synthase protein, is a prokaryote.                                                                                              (29)

Question 2

How will you ensure that the PCR amplicon that you have obtained is the correct gene from devil’s claw? Describe the steps that you will follow in detail and provide all the details of the techniques that you use in each step.                                                                                                                                                              (11)

Question 3

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)

Question 4

Describe all the steps that you would take to clone your gene into E. coli. Draw a diagram of your vector containing the gene insert. Discuss how you will identify a clone in an E. coli cell that you can work further with. Describe the steps that you will follow in detail and provide details of the techniques that you use in each step and how your approach will impact on these steps.                          (25)

Answer 1

ANS (4)-Gene cloning is the process through which gene of interest from the whole genome of an organism is used to make clones(copies) by inserting in a DNA of plasmid vector.

Plasmid vector- Plasmid is the extra circular DNA present inside the e.coli bacterium that has a capacity to replicate independently inside the cell. It is used as a vector because of its self-replicating capacity (because it has own origin of replication), so it can replicate the gene of interest without any problem.

• Polymerase chain reaction is used for cloning the gene and the process occurs in in-vitro condition(In test tube).

PROCEDURE OF GENE CLONING.

1. Isolation of gene of interest.--->Gene of interest is isolated from the whole genome DNA of an organism by cutting out the gene of interest from the whole genomic DNA of an organism using an enzyme restriction enzyme.

• Restriction enzyme will also cut out the small segment of DNA from the plasmid vector DNA and leaves the sticky ends behind it where the gene of interest is going to add on the next step.

2. Ligation---->In this step the gene of interest is attached at the sticky ends of plasmid vector DNA by the help of an enzyme ligase.

• Gene of interest + Plasmid vector DNA ------> Recombinant DNA

3. Transformation--->In this step plasmid vector carrying the gene is interest in transferred into the e.coli bacterium by the process called transformation. The Plasmid along with the gene of interest gets replicated inside the E.coli bacterium so on every cycle of replication the amount (number) of the plasmid vector will increase and that will also cause to increase in the amount of gene of interest with it.

4. Screening(selection)process-----> After the transformation process the thousands or more bacterial cloned cell will obtain in which some of them carry a gene of interest and others were carry only the plasmid DNA, so the selection only for those clones that carry the gene of interest is occurred by various techniques and this process of selection is called screening process.

Cloned gene will be identified blue-white screening technique.-

• In this technique a lacz gene which is also known as reporter gene is inserted in the plasmid vector DNA, this gene encodes for beta-galactosidase.
• The beta-galactosidase enzyme is responsible for breaking a synthetic substrate named as X gal into a soluble blue coloured product
• If a gene of interest is inserted into lacZ , this gene will get in inactivated form and this causes no production of blue colour instead of that white colour appears.
• The host cell having a gene of interest (recombinant DNA) forms a white coloured substrate on a medium having X gal substrate.
• Now on the basis on the colour selection of recombinant cell will obtain as the white colonies having gene of interest (recombinant DNA) and the blue clones did not have the gene of interest.