Question

You have isolated a strain of brewer's yeast (Saccharomyces cerevisiae) in the lab for your second job at a hip new microbrew (you make a great IPA). You find that this strain ferments more efficiently and adds a superior flavour profile to your brews. You want to clone the strain of yeast and generate a genomic library to determine the genes responsible for this finding. To do so you: 1. Obtain fragments of the whole yeast genome (DNA) through restriction digestion 2. Open your vector and ligate the DNA fragments 3. Transform E. coli with these vectors 4. Select for E. coli that have obtained vectors 5. Screen for E. coli transformed with recombinant plasmids 6. Obtain recombinant plasmids from the library and transform yeast with them 7. Clone by complementation the gene that can give superior fermentation. You use the cloning vector pBlue to generate your library. For each of the listed regions on pBlue list the steps in your plan above (1-7) for which the sequence is required and explain why b1) Ori (yeast): b2) Ampr b3) Ori (E.coli)- Or lacz pBlue T Cloning site Ori (E. coli) Note: the cloning site is within the lacZ gene.

Answer 1

Solution

b1) Ori yeast is required for step 7 whree cloning of plasmid vector done inside yeast.

where replication of plasmid is carried out by Ori yeast site to make number of plasmid copies.

Ori yeast is site of initiation of replication in yeast cell of plasmid DNA.

b2) Amp resistant gene is required to select the E Coli in step 4.because

it shows whther plasmid is transformed or not inside e coli.where e coli are cultured in medium containing ampicillin.bacteria having transformed by plasmid vector are remain living and those that dont have plasmid vector will die because they dont have resistant gene of Amp.

b3) Ori E coli-is required for stem 4 and 5.because for selection and screening of e coli plasmid vector and recombinant plasmid vector there is requirment of origin of replication site on both vectors for cloning. Without this site copies of vector can not be done.