You are working in a lab and your goal is to clone the gene for human insulin, s

Question

You are working in a lab and your goal is to clone the gene for human insulin, shown as a black box on the chromosome below. The laboratory has three types of bacterial plasmid vectors that you can use for your experiment labeled I, II and III, each containing the ampicillin resistance gene (amp') and a selectable marker, the lacZ gene. Each vector has different restriction sites engineered within the lacZ gene (shown on the right-hand side of the plasmid). Grown in the presence of the chemical XGal, cells that are lacZ+ will turn blue. XGal does not harm bacteria cells in any way 2. sites in plasmid Restriction map of insulin gene dector Sall Ndel EcoRI Ndel Ndel Afel Sall Insulin gene Ndel EcoRI Vector II Sall amp aVectr EcoRiVector II BamHI amp DNA restriction sequences (The cutting site for each enzyme is indicated with both/and an arow) S'GAATTC 3'CTTAA/G5 NdelS'CA/TATG3EcoRl 3'GTATIACS BamHl 5'G/GATCC3 Sall 3'CCTAGAG5 Afel 5'AGCGCTS (a) What is/are the BEST POSSIBLE enzyme(s) (or combination of two enzymes) to use to excise the insulin gene from the chromosome? Please EXPLAIN. (0.5 pts.) (b) which plasmid vector (1,T1 or 111) would you choose to make the recombinant plasmid and why? (0.5 pts.) (c) What would be the genotype for ampicillin sensitivity and for the LacZ gene of the E. coli cells you would use to clone your insulin gene? (0.5 pt.) (d) Indicate the correct order in which you would perform the following steps in order to transform your E. coli cells with the insulin gene (0.5 pts.) transform E. coli with electroporation or CaC12 cut DNA and plasmid with restriction enzymes select for cells containing the plasmid with the gene inserted add DNA ligase mix DNA and plasmid

Explanation / Answer

a. Sal I or Sal I and Afe I combination

b. SalI single digestion releases Insulin gene. It can be cloned into SalI digested Vector-II

c. The transformed E. coli cells will be Amp +ve and LacZ negative.

d.

i. Cut DNA with restriction enzymes

ii. Mix DNA and plasmid

iii. Add ligase

iv. Transform cells

v. Select positive clones

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