Question 2 continued d) You next transform the E. coli bacterial cells with a mi

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Question 2 continued d) You next transform the E. coli bacterial cells with a mixture of plasmid and the Gene Z cDNA that you have digested with the restriction enzyme(s) that you selected in part (c), plate them on specific medium, allow the bacterial cells to grow and form colonies and tabulate the results belovw - Tranformedcolonies/ugorDNA Media +Ampicillin Media one Gene Z cDNA and Plasmid digested with1.1X 10 40 restriction enzyme that you selected in part (c) Ligation mix of Gene Z cDNA and Plasmid 2.1x10 150 digested with restriction enzyme that you selected in part (c) Based on the number of colonies obtained in Row 2 of the table above, give the phenotype of the E. coli cells prior to transformation. i. Although the E. coli cells in Row 3 of the table above have been transformed in the absence of ligase, you still observe a significant number of colonies. Why is this so? ii. nant ii. In Row 3 of the table above, do you expect all the transformed colonies to contain recombi plasmid that has GeneZ cDNA insert? Why or why not? e) You next select two transformed colonies (Colony A & Colony B) that have the Gene Z cDNA insert. You realize that the gene could have been inserted into the plasmid in two different orientations. So you isolate the recombinant plasmids from two colonies, digest them with restriction enzyme M resolve the digested fragments by DNA gel electrophoresis, and obtain an approximate profile of the DNA as shown below. Of the two bacterial colonies, which one will express Gene Z? Explain your choice. Note: You may assume that the restriction enzymes sites in MCS are only a few bases apart and the MCS is

Explanation / Answer

d) i) the E.coli cells are ampicillin sensitive prior to transformation and cannot survive on media containing ampicillin. However very few bacterial cells are ampicillin resistant, that gave rise to the five colonies observed in the absence of any plasmid DNA.

ii) A significant number of transformed clones observed in the absence of ligase can be due to incomplete digestion of the plasmid. The intact plasmid, left after restriction digestion transformed the bacterial cells and made them ampicillin resistant.

iii) No, all the transformed clones does not contains the plasmid with the insert, it can have clones harboring self ligated plasmid, which lacks the insert. The plasmid backbone can self-ligate without the gene of insert and if transformed into bacterial cells, can impart ampicillin resistance to them.

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