QUESTION 2 (10 marks) Recently, you found out that, there is a jellyfish variety
ID: 183225 • Letter: Q
Question
QUESTION 2 (10 marks) Recently, you found out that, there is a jellyfish variety that can only be found in Malaysia that has blue fluorescence (BF) gene. You wanted to clone this BF gene into a pBR322 vector (Figure 1). The size of the vector is 4363 bp and the BP gene size is 1750 bp. This vector has EcoRI, Bamil, Sall, Prud and Pstl recognition site. The recognition and cleavage sites for these enzyme are EcoRI-GAATC(Banlil G GATOS GTCGAC, pv1 = CTAG,CG and Psrl-CTGCAO. This jellyfish genomic DNA has been digested by Saus A enzyme ('GATC) 4763 Pstt tet pBR322 orl Ndel Figure 1 a) Choose and decide the most suitable site to insert the BP gene in pBR322 vector Justify your decision. Decide the screening strategy to identify the positive clone that carries the correct BP gene insert and justify your decision. What is the expected size of the recombinant vector harboring the insert? Show your calculation. b) e) You wanted to transform E.coli competent cells with this new recombinant vector by the heat-shock method. Discuss the three (3) important steps during heat shock transformation procedures d)Explanation / Answer
a) The most suitable site for inserting BP gene in pBR322 vector is BamHI with GGATCC. The digestion of vector with BamHI enzyme will generate -GATC overhangs which are compatible to the overhangs generated by Sau3A enzyme (It also genertaes -GATC overhangs). So, the DNA fragments generated by Sau3A enzyme can be ligated into vector cleaved by BamHI enzyme.
b) The positive clone that carries BP gene insert is resistant to antibiotic ampicillin and sensitive to tetracycline. The positive clone will be sensitive to tetracycline because BP gene is inserted in BamHI site present in tetracycline gene. Positive clones carrying BP gene will grow on ampicillin containing medium but not grow on tetracycline containing medium. Clones containing pBR322 plasmid without BP gene can grow on both ampicillin and tetracycline containing medium. In this way positive clones carrying BP gene can be screened from clones carrying only pBR322 plasmid without any insert.
c) The expected size of the recombinant vecor harboring the insert is 6113 bp. The vector size is 4363 bp and insert size is 1750 bp. So, 4363+1750=6113 bp.
d) Step 1) Thawing of competent cells on ice, addition of recombinant vector to competent cells and incubation in ice for 30 minutes.
Step 2) Heat shock cells at 94 degrees centigrade for 45 seconds and immediately transferring to ice.
Step 3) Addition of growth medium to the cells and incubate at 37 degrees centigrade for 90 minutes.
After 90 minutes, cells were plated on antibiotic containing growth medium to screen positive clones containing gene of interest in the vector.