2016 VJC H2 Biology Prelims Paper 3 Answers

1 Question 1 (a) The bacterial plasmid, pBR322, was used as a vector for Gene X as shown in Fig. 1.1 below. Gene X was inserted in the BamHI restriction site. pBR322 also contain the EcoRI and HpaI restriction sites. The target sites for these restriction enzymes are shown in the table below. The lines drawn in each sequence show where the enzyme cuts the DNA molecule.     (i) With reference to Fig. 1.1, explain how two properties of plasmid pBR322 allow it to be used as a vector. [2]  Presence of origin of replicati on; so inserted gene can be replicated;  Has ampicillin and tetracycline resistanc e genes; as selection markers / allow identification of host cells that have successfully taken up the recombinant plasmid (ii) Outline the steps taken to produce the recombinant plasmid shown in Fig. 1.1. [2]  Cut plasmid and gene X with BamHI restriction enzyme;  “Sticky ends” generated;  Allow plasmid and gene to anneal wit h ref. complementary base pairing; 2016 H2 Biology Prelim Paper 3  ANSWERS

2  Add ligase to seal the nicks in the sugar-phosphate backbone / form phosphodiester bonds between the ends of the cut plasmid and gene; (iii) Explain the disadvantage that would arise if gene X was to be inserted into the HpaI restriction site instead of the BamHI site. [2]  HpaI generates “blunt ends”;  No hydrogen bonds to hold t he cut plasmid and gene together;  Ref. extra step that will require linker;  To generate “sticky ends” (b) Calcium chloride heat shock treatment wa s then used to introduce the recombinant plasmid into Escherichia coli . However, the process of creating recombinant plasmids is typically not 100% efficient. Often, a mixture of re-annealed plasmid and re-annealed DNA is produced along with the recombinant plasmid. These may be taken up by the bacteria as well. This nece ssitates the process of selecting for the bacteria that have successfully taken up t he recombinant plasmid. As such, the bacteria was first plated onto a nutrient agar plate containing ampicillin. Replica plating was subsequently carried out onto a nutrient agar plate containing tetracycline. Bacterial growth on both plates is shown in Fig. 1.2. With reference to Fig 1.2, (i) Circle the colonies that were successf ully transformed with the recombinant plasmid. [1] (ii) Account for the difference in colony numbers in Plate A and B. [3]  Plate A selects for all successfully transformed cells/ taken up plasmid with ampicillin gene; (maybe recombinant plasmid or re-annealed plasmid);  Selected against / killed off cells which took up re-annealed DNA;  Plate B contain 3 fewer col onies compared to Plate A;  Missing colonies had successfully ta ken up the recombinant plasmid;  where gene X had been inserted into tetrac ycline gene and disrupted it (ref. to insertional inactivation) / colonies lost tetracycline resistance and died;

3  Therefore