Question

Ocean pout, a fish found in cold waters, produce an antifreeze protein that allows them to survive at near freezing temperatures. Your company plans to genetically engineer bacteria to grow antifreeze proteins to improve the storage of ice cream products

a) The vector and PCR product that you will use to clone the antifreeze gene are shown above along with the locations of three different restriction enzyme cut sites Which restriction enzyme (Hindill, BamH1, or EcoR1) will allow you to successfully clone this transgeno? Brietly justify your answer.

b) Describe how you will select for bacteria that carry the recombinant DNA molecule once you have completed the transformation

Answer 1

A) BamH1 restriction enzyme is used to cut the vector and gene of interest. This will allow succesful cloning of the gene. Because the BamH1 gene is found outside the coding region of gene in gene of interest. And it is also present in ampar region in the plasmid. This further helps in selection of recombinant bacteria. B) You can put both kind of bacteria in presence of UV light. You will find that recombinant bacteria will glow in presence of UV because of GFP protein. But the non recombinant bacteria will not glow. In this way, you can isolate the recombinant bacteria. other method, but not so much useful: The bacteria is grown in presence of ampicilin containing media. The bacteria which has recombinant plasmid will not grow in presence of ampicillin containing media because ampar gene is non functional in recombinant bacteria because of insertional inactivation. Where as other untransformed bacteria will grow. So,first method is best.